Stanford University

NIH Research Projects · FY 2025 · 2021-03

Project Summary The gut microbiome exerts a tremendous influence on the function of the immune system at homeostasis and during inflammatory insults. Most research has focused on how individual bacteria or particular bacterial configurations interact with the immune system, but the microbiota contains other forms of microbial life, including protists. Several species of common and non-pathogenic protists in mice, from the genus Tritrichomonas, induce a type 2 immune response in the small intestine without causing overt pathology. In addition, other species of Tritrichomonads were later discovered that promote Th1 and Th17 cells in the colon. However, it is unclear if these differences in mucosal immunity are related to species- specific effects or other factors. The phylogenetic relationship between intestinal Tritrichmonas species is poorly understood as their genomes remain unsequenced. Furthermore, the immune landscape and microbiota composition varies greatly between the small intestine and colon, which further complicates comparisons between these Tritrichomonas species and their immune effects. To overcome these obstacles, we isolated two species of Tritrichomonads that elicit either a Th1 or Th2 immune response in the small intestine. In Aim 1, we will generate high-quality genomes for each protist and measure their output of key immunomodulatory metabolites. In Aim 2, we will characterize the effects of these two protists on the adaptive immune system. We will determine immune mechanisms that respond to each species of Tritrichomonas. Finally, in Aim 3, we will determine how these two Tritrichomonads impact Crohn’s Disease in a mouse model. These studies will establish key genomic tools and insights into Tritrichomonas species and mechanisms that influence on intestinal immunity. Furthermore, this project will evaluate the possible benefits of these symbiotic protists on Crohns’s disease, leading to novel therapeutic approaches to treat IBD.

NIH Research Projects · FY 2025 · 2021-02

Imaging and modulation of immunophenotype Most importantly, we have created TLR delivery nanotherapeutics and find that TLR NPs combined with aCD40, aPD-1 and aCTL4 (abbreviated as CP4 as in emerging pancreatic cancer studies) efficiently regressed implanted multisite invasive murine pancreatic tumors. We have developed multiple strategies and are particularly focused on 18 nm biodegradable, multi-functional particles that combine immune-modulating peptides, targeting peptides and toll-like receptor (TLR) agonists. We specifically included the immune modulating peptide PADRE (T helper modulation) and mannose (to enhance macrophage uptake) in addition to the TLR7/8 agonist (resiquimod) in preliminary work. To maximize payload, we built upon biocompatible unimicellar nanoparticles via the combination of highly efficient esterification and metal-free click reactions and find that the particle metabolites clear through the kidneys. In our preliminary studies, TLR7/8-nanoparticle treatment combined with CP4 enhanced response in a highly metastatic, multi-site implanted pancreatic cancer model (Kras+/LSL-G12D; Trp53+/LSL-R172H; Pdx1-Cre model: abbreviated as KPC). New preliminary data indicate that TLR7/8 agonists and aCD40 each have direct efficacy against pancreatic tumor cells. RNAseq results demonstrate that TLR7/8 agonists and CD40 enhance complementary pathways (C-lectin for CD40 (among others) and TLR/interferon for TLR agonists). We find that the combination enhances anti-tumor leukocytes, regresses KPC tumors and for responders, 100% do not grow tumor on re-challenge. By monitoring OX40 expression (a marker of T cell activation), we demonstrated that unlike other immune modulating approaches involving aCD40, T cells were activated. We have simultaneously developed the ability to monitor OX40 expression using positron emission tomography in a noninvasive fashion. Our primary goal in the proposed work is to move the nanotherapy strategy forward to human translation. As a result, we will evaluate efficacy in models of pancreatic cancer in rodents and safety in a larger animal model. Further, we will evaluate samples from patients undergoing biopsy for pancreatic cancer to better characterize the immune environment. We have 2 major goals: 1) the development of an effective strategy for systemically-administered T cell modulation and 2) combining this with positron emission tomographic imaging and RNA sequencing to optimize multi-component protocols. Within Aim 1, we will determine the optimal carrier properties to maximize T cell modulation by 1a) modulating nanoparticle characteristics and evaluating resulting efficacy, 1b) using positron emission tomography (PET) imaging to quantify accumulation of the systemically-injected NP agonists, and 1c) assessing toxicity through dose escalation and a large animal study, leading to IND filing. Within Aim 2, develop an imaging and in vitro assessment strategy for T cell activation by utilizing 2a) OX40 PET imaging and 2b) flow cytometry and RNAsequencing.

NIH Research Projects · FY 2025 · 2021-02

ABSTRACT / PROJECT SUMMARY HLA-B*51 in epistasis with the ERAP1 haplotype Hap10 confers the strongest currently known risk for Behcet’s Disease (BD) and Behcet’s Eye Disease (BED), but the consequences of this relationship for immune-phenotype, clonality, and function, are entirely unknown. The main objective of this application is to identify these consequences. This is in line with our long-term goal to find, comprehend, and correct aberrancies in immune pathways that drive Behcet’s Eye Disease (BED). Based on our preliminary studies we propose as our central hypothesis that ERAP1 Hap10 shapes immune responses in HLA-B*51+ BED through the activation of clonal CD8 T and NKT effector cell populations that drive the disease. We follow the rationale that elucidation of the biological consequences of HLA-B51/ERAP1 Hap10 will promote a mechanistic understanding of BED in affected carriers, and therefore enable targeted therapy design. We will test our central hypothesis in two specific aims: 1) Through the determination of immune phenotypes linked to HLA-B*51+/ERAP1 Hap10 BED, combining access to unique patient cohorts of our own and those of our international collaborators with large-scale flow cytometric analyses using computational tools we have developed. 2) Through the identification of clonal effector responses and function in BED patients using single cell-transcriptomics and methods of T cell cloning we have established. Meeting the objective of this proposal will generate knowledge providing scientific rationale for the design of targeted therapies for BED, which is one of the most devastating forms of non-infectious uveitis with significant prevalence in large parts of the world. We expect additional positive impact by cross-fertilizing research of other HLA-I/ERAP-related diseases including HLA-B27-associated uveitis and spondylitis, Birdshot’s choroidopathy, psoriasis-associated conditions, and inflammatory bowel disease (IBD).

NIH Research Projects · FY 2025 · 2021-02

Project Abstract Overview: In 2020, over 276,000 women will be diagnosed with invasive breast cancer, and over 48,000 will die from it. The ultimate goal of this project is to provide fast, accurate, accessible non-contrast-enhanced MRI screening methods to safely detect breast cancer in the high-risk population. Relevance: Screening mammography is successful in reducing breast cancer mortality, but misses many important cancers, especially in women with a high risk of breast cancer, and in the 27 million women with dense breasts who undergo screening. MRI has been shown to be more than twice as sensitive for screening in many scenarios, but high cost, discomfort, and poor compliance due to the need for IV gadolinium-based contrast agents with side effects limit its impact for screening. This project aims to change this paradigm by developing a rapid, inexpensive, comfortable, non-contrast-enhanced breast MRI screening exam. Approach: Based on substantial prior work, our group will develop high-resolution 3D and 2D diffusion MRI methods that can depict important morphologic features and quantify diffusion heterogeneity in breast lesions. To support these methods, we will first implement flexible, closely-fitting soft breast coil arrays that maximize SNR and parallel imaging capabilities while reducing positioning time and increasing comfort. We will combine the efficiency and motion-insensitivity of non-Cartesian 3D cones imaging with the double-echo steady-state dif- fusion approach to offer efficient 3D diffusion-weighted imaging (DWI). Additionally, we will redesign echo-planar imaging (EPI) DWI including asymmetric encoding with bulk motion insensitivity, full k-space readouts, and novel locally low-rank reconstruction to provide high-resolution quantitative DWI that is robust to small patient motion. Finally, collaborating with two other major centers, we will study these methods in high-risk screening patients, aiming to demonstrate improved non-contrast-enhanced sensitivity from 45% to over 70% among women with negative mammograms, while retaining specificity over 91%. If successful, these rates are sufficient to make non-contrast-enhanced MRI screening viable for clinical use. Summary: Using advances to 2D and 3D DWI, combined with flexible coil arrays, we aim to provide accurate, low-cost, comfortable, MRI screening without intravenous contrast, in a 10 minute exam. This will ultimately en- able more effective and comfortable breast cancer screening for millions women for whom x-ray mammography is insufficient.

NIH Research Projects · FY 2025 · 2021-02

PROJECT SUMMARY Triple-negative breast cancer (TNBC) is the deadliest and 2nd most common subtype of breast cancer in the United States. Although promising new drugs based on PARP inhibition and immunotherapy can extend survival in selected patients, 1 in 3 patients die from TNBC. Increasing evidence suggests that human breast tumors harbor immature cancer cells which are a distinct subset of tumorigenic cancer cells, are less-differentiated, capable of replenishing cancer cell populations indefinitely, and strongly implicated in drug resistance. Unfortunately, existing marker genes for studying these cells are not specific, precluding rational drug development. We hypothesize that precise identification of immature cancer cells could present new therapeutic opportunities to revolutionize TNBC treatment. We recently showed that the number of expressed genes per cell is a powerful surrogate of cellular differentiation status independently of known marker genes. We leveraged this finding to develop CytoTRACE, a new framework for predicting cellular differentiation status from single-cell RNA sequencing (scRNA-seq) data. Our published data show that immature cancer cells predicted by CytoTRACE preferentially express genes essential for tumorigenicity in TNBC. In pilot data, we identified 10 putative cancer cell populations, including at least 3 immature ones, from scRNA-seq data of 19 primary breast tumors. Here, we propose to study over 800 TNBC patients to determine whether immature cancer cells represent at least 3 distinct populations (Aim 1); differ by key clinical covariates (Aim 2); and are clonogenic and produce specific progeny populations predicted in silico (Aim 3). To accomplish these aims, we will leverage new analytical methods, including a deconvolution approach for integrating scRNA-seq with bulk tumor transcriptomic data in order to characterize cellular heterogeneity at scale. Successful completion of the proposed project will validate and refine our pilot data toward advancing our understanding of cancer cell populations, especially immature cells, in TNBC. As such, we expect this study to facilitate new opportunities for the development of targeted drugs to improve TNBC outcomes.

NIH Research Projects · FY 2025 · 2021-01

PROJECT SUMMARY/ABSTRACT Over the last twenty years, an increasing number of agricultural communities have faced an apparently new, unexplained, and fatal kidney disease, known as chronic kidney disease of unknown etiology (CKDu). First noted in sugar cane workers in El Salvador and rice farmers in Sri Lanka, reports of a similar kidney disease have emerged come from Nicaragua, Costa Rica, Guatemala, India, and (most recently) the U.S. Despite the scale and severity of this kidney disease epidemic, the epidemiological and mechanistic investigations needed to address it have been extremely limited. Because persons with the disease are otherwise healthy agricultural workers, many experts and the affected population suspect agrochemical exposure is responsible. In two key preliminary studies from Sri Lanka, we find that agricultural workers are drinking from shallow water wells that are contaminated by organophosphate and organochlorine agrochemicals above EPA drinking water regulations, and well water consumption raises likelihood of biopsy-proven CKDu and faster progression of established kidney disease. In a cohort of 600 at-risk participants identified by our preliminary work in whom we will obtain baseline environmental samples including water samples and kidney biopsies if they meet a validated clinical definition of CKDu, we propose to examine the hypothesis that specific agrochemicals contaminating well water are causing CKDu. We will: 1) run untargeted and targeted mass spectrometry analysis of well water, 2) determine the association of individual agrochemicals and their mixtures with incident CKDu case status, accounting for work intensity and heat stress, 3) measure the bioburden of nephrotoxic agrochemicals in cases versus controls, and 4) perform molecular analyses of early-stage kidney biopsies to specify the injury response pattern at a cellular level with bulk and single-cell RNA sequencing. In alignment with NIDDK-NIEHS-Fogarty recommended approach to CKDu investigations, this proposal integrates a multi- disciplinary, multi-national team of nephrologists, pathologists, molecular biologists and environmental geochemists. Based on our preliminary data we focus on agrochemical exposure via well water as the environmental risk factor of interest in this proposal, however field work will be coupled with an extensive biobanking effort to facilitate testing of multiple candidate hypotheses. The complementary molecular analyses will precisely characterize the injury in CKDu in the context of other primary tubulointerstitial kidney diseases, and create a rigorous scaffold for testing potential agents that can trigger CKDu-specific responses in the kidney. As in the case of prior regional kidney disease epidemics such as Balkan nephropathy, the intensive effort to identify cause in our outlined aims has the potential to pinpoint other vulnerable populations and regions, and more importantly, to abrogate the kidney disease by eliminating the exposure.

NIH Research Projects · FY 2025 · 2021-01

Abstract Frequent, accurate, and highly sensitive HIV-1 viral load monitoring is a critical component of AIDS antiretroviral therapy, a tool for reducing the incidence of mother-to-child HIV transmission, and a required element of routine diagnostic testing to make people aware of their HIV status. Although enormous research and product development effort has been applied to point-of-care viral load testing, the current paradigm of nucleic acid tests and antigen assays continues to demonstrate fundamental limitations that derive from their inherent complexity and lack of robustness, which in turn impact their costs and practicality for adoption in resource-limited settings. We seek to address an important gap in the capabilities of existing technologies through a combination of three innovations to yield an integrated, rapid, simple, ultrasensitive, highly selective, robust, and inexpensive system for quantitative viral load measurement. First, we utilize microfluidic separation of virions from whole blood, yielding a 10-50 µl plasma sample from 20-100 µl of whole blood in <10 min, with >95% virus extraction efficiency. Second, we will achieve ultraselective recognition of intact HIV virions from the resulting serum using designer DNA nanostructures that take the form of a macromolecular “net” whose vertices are a precise mechanical match to the spacing and positioning of the spike gp120 protein matrix displayed on the HIV outer surface. The DNA net vertices incorporate nucleic acid aptamer probes that have been selected for selectively targeting the HIV gp120, resulting in multiple sites of high affinity attachment, and thus the “net” can be used as an effective capture probe when covalently attached to a photonic crystal biosensor surface. Finally, we will utilize a newly-invented form of biosensor microscopy called Photonic Resonator Interference Scattering Microscopy (PRISM) in which the photonic crystal surface amplifies laser light scattering from captured intact virions, enabling each one to be counted with high signal-to-noise ratio. Because PRISM does not require labels or enzymatic amplification, our approach enables dynamic, real-time counting of captured virus with digital precision and ultrasensitivity. In the proposed project, we will integrate viral separation and the photonic crystal biosensor into a plastic cartridge and develop a rapid workflow that will be simple and rapid for compatibility with point-of-care settings, with the goal of yielding a result in <30 minutes sample-to-answer. Our Aims include development of a point-of-care version of the PRISM instrument, and statistically robust characterization of detection limits, repeatability, and robustness. Our study will conclude with validation of the system using clinical specimens and direct comparison against gold-standard laboratory RT-PCR analysis.

NIH Research Projects · FY 2025 · 2021-01

PROJECT SUMMARY Vision is an active sense, with eye movements powerfully shaping the acquisition of visual information about the world. This project investigates how motor learning adjusts the neural circuitry controlling eye movements, to maintain the accuracy of eye movements over short and long time scales. The specific focus of the research is to understand how oculomotor learning, i.e., improvement of the accuracy of eye movements through experience, is transferred from short-term to long-term storage. This consolidation process occurs, not only for motor skills like eye movements, but is a general feature of learning and memory systems. Some memories, including oculomotor memories, are transformed during the time after the initial acquisition of the memory, in a way that renders older, consolidated memories independent of brain areas that are critical for newer memories. This process, known as systems consolidation, is thought to depend on activity of neurons in the brain area initially critical for the memory, and the hypothesis is that this activity induces changes in the brain area(s) supporting long-term storage of the memory. The proposed research characterizes the nature of the neural signals transmitted between brain areas supporting memory at different times after their acquisition, and the rules that operate on those neural signals to implement stable transfer of a memory from one brain area to another.

NIH Research Projects · FY 2026 · 2021-01

PROJECT SUMMARY Cells and organisms, from simple to complex, carry the same genetic DNA sequence in each cell. This identical DNA blueprint gives rise to cells with remarkably different, specialized functions because RNAs expressed from the DNA have dramatic differences between cells. Much of is expressed variation is due to sequence diversity and difference of RNA transcripts from the same gene that perform different functions. Multiple processes act to generate transcript diversity that is only partially captured– and therefore only partially understood– by the current way genomic data is analyzed: through genome assembly and alignment. For example, we discovered circRNA being a ubiquitous product of alternative splicing only in 2012, and its regulation and function remains enigmatic. circRNAs’ discovery revealed a larger critical knowledge gap in the field for “what, how and why” different transcript variants exist. Examples of transcript variation arise from critical processes from RNA splicing to editing to V(D)J DNA recombination that generates the adaptive immune system that enable diverse functions from the same genomic locus. Today, most of these diverse transcripts have unknown functions; in cases where their functions are known, they can have context- dependent actions: acting differently in different cell types or conditions. The sheer number of variants (>100,000 in the human genome alone), and the enormous number of cell states where they could have different functions make this problem infeasible to study experimentally via traditional experimental biology. We propose a radically different computational approach to predicting variant functions through a new approach to sequence analysis, SPLASH. SPLASH flips the analytic paradigm from alignment or interpretation first to a statistics first approach to analyzing sequence data that does not require a reference or any metadata knowledge of the sample origin. This approach increases the speed and throughput of genomic analysis and also increases the sensitivity to discover new transcript variants. We have already used SPLASH to discover new genes and new variants of genes in the human transcriptome. In this proposal, we will build new generations of the SPLASH algorithm, develop new statistical approaches to discover signatures of functional selection in RNA, and together use massive data mining to develop machine and deep learning models to predict how transcript variance are co-regulated and determined cellular phenotype. Answers will also enable a new generation of diagnostics for disease, drug targets for correcting dysregulated splicing and identification of pathogenic protein- or non-coding products (respectively) as well as fundamental basic scientific insight into evolution and function of eukaryotic genomes. The proposed research will couple novel statistical analyses of genomics data by taking an unbiased approach and including biological features that are understudied or un- annotated. Predictions will be coupled with incisive experimental validation to reveal new principles of how RNA variants function and how characterizing them in health and disease can be used for precision medicine.

NIH Research Projects · FY 2025 · 2020-12

Project Summary The objective of PREcision Care In Cardiac ArrEst - ICECAP (PRECICECAP) is to discover novel biomarker signatures of post cardiac arrest brain and extracerebral organ failure that predict treatment responsiveness and long-term recovery. We will achieve this by partnering with the ICECAP trial, a response-adaptive dose finding clinical research trial that seeks to determine the optimal duration of post-arrest hypothermia. Cardiac arrest is a major public health problem with high morbidity and mortality. Four in five patients hospitalized after cardiac arrest have significant brain injury, and death from neurologic damage is common. Improving survival and functional recovery is a critical public health objective and will require innovative approaches. The current clinical situation is unprecedented. Currently post-CA brain injury is an acute, sudden critical illness with major knowledge gaps about how best to characterize severity of injury and to identify which individual patients are likely to benefit from specific neuroprotective strategies. Thus, development of high- performing biomarker signatures is a critical need and would translate in to immediate changes in care. We hypothesize that not all patients are identical (i.e. there will be a heterogeneity of treatment effect) and that through our innovative, multi-parametric data driven approach, we will be able to identify novel signatures that define subgroups of patients. Advanced data science and analytical approaches will allow the identification of these subgroups that was previously not possible. These subgroups will be clinically important insofar as they will indicate differential responses to treatment and different trajectories of functional recovery. This project will acquire high resolution multi-modal data early in the disease course that will allow us to address these current knowledge gaps and improve our understanding of the disease in the early acute setting when interventions can improve outcome. The knowledge learned here will be applied to develop personalized treatments for cardiac arrest survivors, addressing the NIH's and our goals of lengthening life and reducing disability.

NIH Research Projects · FY 2025 · 2020-11

PROJECT SUMMARY/ABSTRACT Diffusion MRI (dMRI), with its sensitivity to neurological changes, has contributed immensely to our understanding of the white matter structure at large-to-medium spatial scales, and more recently towards smaller structures, particularly in the cortical and subcortical regions, as submillimeter dMRI is starting to become more feasible. Nonetheless, further increase in resolution is required to move dMRI to the mesoscopic scale (100- 500µm), to improve sensitivity to small but important brain microstructural features and improve capability to e.g. detect highly-localized tissue abnormalities such as focal cortical dysplasia and silent acute microinfarcts, as well as enable study of brain connectivity across laminar structures and delineate functionally important small sub-cortical gray matter structures. In this proposal, we aim to develop imaging technologies to allow mesoscale dMRI and extend our development to enable mesoscale joint diffusion-relaxometry MRI with rich information, to improve tractography’s robustness and enhance capability to extract detailed microstructural information. Mesoscale dMRI and diffusion-relaxometry MRI will enhance the capability of many current clinical and neuroscientific investigations and open doors to entirely new ones, facilitating new discoveries to deepen our understanding of the human brain in both its healthy and diseased states. In the previous funding cycle, we developed the gSlider-SMS technology to allow 700-800µm dMRI to be acquired in a short 20-minute timeframe and shared it across 26 research institutes world-wide, where it is being used to investigate numerous neuroscientific questions and neurological disorders, including aging, epilepsy, acute micro-infarcts, and deep-brain stimulation planning. For this renewal, we propose to create technologies to allow 300-500µm whole-brain dMRI in 20-30 minutes, with fast reconstruction, suitable for wide-adoption. We will then expand our development to create a joint T2-dMRI acquisition for fiber-specific microstructure imaging and improved fiber tracking capability at the mesoscopic scale. Lastly, we will also create a highly efficient joint T1-T2-dMRI acquisition, to rapidly collect large multidimensional data and provide diffusion-relaxation correlation spectroscopic map per imaging voxel for detailed microstructural investigation. The multitude of imaging technologies that we will develop will enable creation of new richer datasets that will have a significant and lasting impact on the neuroscientific study of the human brain and many clinical applications. Optimized protocols will be developed for high-end clinical 3T MRIs for wide deployment, and on high-performance head-only 3T MRIs to enable neuroscientific explorations at the limit.

NIH Research Projects · FY 2024 · 2020-09

PROJECT SUMMARY / ABSTRACT The long-term objective of the research proposed here is to establish a novel method for creating vaccines. These vaccines will lead to a highly focused antibody response toward particular epitopes that are known to be the targets of neutralizing monoclonal antibodies (mAbs). If successful, this approach could be applied broadly for the creation of important, new vaccines that protect against infectious disease. The ability to focus the antibody response toward particular epitopes would permit vaccines to be created that elicit neutralizing antibodies, instead of non-neutralizing antibodies. It would also permit creation of vaccines that lead to an antibody response directed against highly conserved regions of an infectious agent, leading to broad spectrum protection against different strains and minimizing the possibility of “escape” by mutant variants. The key starting material for the approach is a mAb that is broadly neutralizing against the infectious agent. In recent decades, many potent, broadly neutralizing mAbs (bnAbs) have been isolated and characterized in detail. Some of these bnAbs (for example, that target influenza virus, Ebola virus and HIV-1) have entered clinical trials to test their efficacy in treating infectious disease and/or to determine whether passively infused mAb can prevent infection. Despite major research funding, however, it has generally not been possible to create vaccines that are capable of eliciting antibodies with properties such as these bnAbs. Here, a simple but radically different approach for creating epitope-focused vaccine candidates is utilized that leverages a tool that has been available all along – the mAb itself. First, binding of the mAb is used to protect the target epitope. Next the surface of the remainder of the antigen is modified to render it non-immunogenic. Finally, the protecting mAb is removed, thereby deprotecting and exposing the unmodified, target epitope. The method is called protect, modify, deprotect (or PMD). Ultimately, this high-risk, high-reward proposal could enable creation of vaccines that elicit an antibody response against any given mAb epitope, and only that epitope.

NIH Research Projects · FY 2024 · 2020-09

Deep brain stimulation is approved for a number of neurological disorders. However, the where, when and how to stimulate the brain are still empirically solved. The goal of this proposal is to develop a systematic framework that will enable a better understanding of the effects of deep brain stimulation. We will expand The Virtual Brain (TVB), a computational model we developed, with a data-driven approach taking into account cell-type specific control. The project relies on a tight interaction between experimentation and computational modelling. First, using enhancer-based genetics, we will activate/silence specific cell types with opto- /chemo-genetics while acquiring resting state and stimulus driven fMRI in individual mice. Each brain will be virtualized in TVB and undergo data fitting, validation and inference using Stan, a platform for statistical modeling. Model predictions will then be verified in the same individual mice. As a first step towards an application in pathologies, the cells and parameters that need to be controlled will be predicted in silico to stop seizures in experimental epilepsy, and to restore resting state dynamics and rescue motor behavior in an experimental model of Parkinson's disease. Predictions will then be tested experimentally. The proposed project will allow the generation and testing of hypotheses concerning the control of specific cell types with unparalleled biological relevance, precision, and speed. The main goal is to show that it is possible to drive brain activity with stimulation in a predictable way. Through cell type specific modeling of whole brain function in mice, we aim to achieve a major milestone in the development of a realistic, large- scale, anatomical, biophysical model of the human brain. Through unique expertise and technologies our international, interdisciplinary team has pioneered, we will address the problem of understanding how brain stimulation can be systematically designed for individual patients. RELEVANCE (See instructions): The success of deep brain stimulation in treating movement disorders led to its application to various psychiatric and neurological disorders, in an attempt to treat symptoms as well as to directly improve memory function. However, recent clinical trials failed to demonstrate significant effects in epilepsy, depression and dementia, calling for new scientific developments to better understand where, how, and when to stimulate the brain to obtain specific effects. Our experiments will provide fundamental mechanistic insight into how the stimulations impact the brain and how it can be designed for optimal therapeutic outcome in individual brains.

NIH Research Projects · FY 2025 · 2020-09

Abstract Novel neuroimmune disorders defined by the presence of autoantibodies in plasma and/or CSF have recently been discovered, most often first described in the context of paraneoplastic syndromes, only later to be also found unassociated with tumors. These include limbic encephalitis (e.g., anti-NMDAR, anti-AMPAR, anti- GABABR, anti-LGI1, anti-CASPR2), cerebellar ataxias (e.g., anti-Yo, anti-DNER, anti-GAD), stiff-person syndrome (anti-GAD, anti-amphiphysin, anti-GlyR) and others (anti-Hu). Although little is known regarding the pathophysiology of these disorders, some are believed to be more B-cell or T-cell mediated, hypotheses mostly suggested by observed therapeutic responses and the surface/intracellular nature of antigens. Involvement of KIR and HLA is unexplored in these disorders. Intriguingly, our preliminary data support a strong Genome-Wide Association Study (GWAS) significant association of anti-NMDAR encephalitis with activatory KIR2DS1, the strongest KIR association ever reported, suggesting KIR-NK cell axis to be a key regulator for these disorders. We propose to conduct a genetic survey of the HLA and KIR repertoires in these diseases. Our specific aims 1 and 2, we will do a detailed characterization of symptoms and collect serum and DNA on 2000 cases (~1000 cases already collected). Our specific aim 3 will leverage state of the art advances in KIR and HLA next- generation sequencing to perform high resolution genotyping in cases and controls, in addition, we will also perform genome-wide single nucleotide typing in all cases and controls which will guide our association analysis. In our specific aim 4, we will perform association analyses of the genome wide genotyping, compare frequencies of HLA and KIR alleles in cases and controls. In our specific aim 5, any disorder with KIR and/or HLA finding(s) we will conduct KIR-HLA interaction analyses focusing on KIR with known HLA ligands. This will involve comparing frequencies of interacting KIR-HLA ligand pairs in cases versus controls; further, we will correlate KIR/HLA findings with disease severity or symptom clusters. The study of these disorders will benefit neurology, neuroimmunology, cancer, and infectious disease work. The fact these disorders are occurring in the setting of heightened tumor immunity is notable and relevant to cancer immunotherapy, a growing area. These datasets will be made available through ImmPort, which will allow countless researchers to prioritize basic studies of B cells, T and NK cells in these disorders and associated tumors.

NIH Research Projects · FY 2024 · 2020-09

Prefrontal cortex (PFC) is critical for a range of high-level cognitive functions, such as attention and decision- making. Studying these processes is difficult, since they are covert, dynamic and under the control of the subject, rather than the experimenter. Their measurement traditionally relies on inferring their presence via behavior. Recent technical advances, particularly the increase in the number of neurons that can be simultaneously recorded, has raised the possibility of decoding cognitive processes directly from neural activity. In the proposed project, we will capitalize on the recent development of high-density, high-channel count, silicon probes (Neuropixels) that can produce a more than 20-fold increase in neuronal-recording yield over conventional methods. We will use these new probes and decoding algorithms to identify the cellular and circuit- level mechanisms of attentional control and decision-making in primate PFC. We will perform this decoding in real-time and use the output to control the application of stimulus perturbations or electrical microstimulation, which will ‘close the loop’ and determine the necessity of the decoded signals for cognition. Finally, we will study the contribution of specific subpopulations of neurons by restricting the decoder to those populations. The first aim focuses on understanding how attentional control is achieved by lateral prefrontal cortex (LPFC). We will record simultaneously from large populations of neurons within the frontal eye field and area 46 of monkeys performing a selective attention task. Neurons will be characterized by the laminar location, by their sensory, motor and memory-related properties, and as putative pyramidal or interneurons. The contribution of specific subpopulations to attentional control will be determined by building a decoder to report the animal’s current attentional locus using neural activity from the subpopulation of interest. In turn, the decoder will be used to control stimulus perturbations to assess the behavioral effect of the decoded signals on attention. In the second aim, we will examine the contribution of orbitofrontal cortex (OFC) neuronal subpopulations to value- based decision-making, including the contribution of different cortical layers, cell types, and OFC subregions. In addition, the decoder output will be used to control the application of electrical microstimulation to examine whether choice behavior can be biased towards a specific choice alternative. In the last Aim, we will build on the work from the first two aims to determine how value and attention interact. While some evidence suggests that attention is prioritized to stimuli with high value, other evidence suggests that the opposite is true. Results from Aims 1 and 2 will be used to optimize a decoder in OFC that will output the value of objects currently under consideration, and a decoder in LPFC that will output the current location of covert spatial attention. We will then cross-correlate both decoder output signals to determine whether one reliably lags or leads the other. The proposed work leverages the expertise of the three PIs: Moore is an expert in the role of LPFC in attention, Wallis in the role of OFC in decision-making, and Shenoy in real-time decoding and closed-loop control.

NIH Research Projects · FY 2024 · 2020-09

PROJECT SUMMARY/ABSTRACT Myelin—the electrical insulator around neuronal axons—is essential in vertebrates for rapid nerve signaling, and its loss in diseases like multiple sclerosis and following injury causes severe disability in patients. In the central nervous system, oligodendrocytes build myelin by first extending their membrane processes to ensheath axons, then wrapping spirally around the axon while compacting their membranes to become electrically insulating. In chronic multiple sclerosis lesions, oligodendrocytes ensheath axons but fail to wrap, suggesting that wrapping is a rate-limiting step for remyelination. To ultimately understand why remyelination fails in multiple sclerosis, we first aim to understand the mechanism by which myelin wraps normally. It was long hypothesized that the assembly of actin filaments provides the force required to drive wrapping, like the lamellipodium of a motile cell or a neuronal growth cone. However, we and others recently discovered that the dramatic disassembly of the oligodendrocyte actin cytoskeleton is required for wrapping. This finding was completely unexpected and suggests two models for wrapping. Cycles of actin disassembly and reassembly could be required to “ratchet” the oligodendrocyte membrane forward. In contrast, based on our preliminary data, we propose that actin disassembly acts as a “trigger” to initiate actin-independent wrapping and that the major role of actin disassembly is to allow myelin to compact. To test these models, we are using a suite of innovative approaches including first-in-class genetic tools we created to experimentally induce actin disassembly (DeActs) or block actin disassembly (StablActs) in oligodendrocytes during wrapping in vivo, advanced microscopy techniques to resolve myelin in vivo, and live cell imaging of oligodendrocytes in culture. Our preliminary data demonstrate: (1) actin filaments disassemble in oligodendrocytes prior to wrapping, (2) experimentally inducing actin disassembly specifically in oligodendrocytes in vivo increases myelin wrapping, and (3) experimentally blocking actin disassembly impairs myelin membrane compaction in a culture model of myelination. These data support the “trigger” model of myelin wrapping, laying the foundation for future translational studies to test whether this actin disassembly-based mechanism is recapitulated or perturbed during remyelination. By defining the role of actin disassembly in myelin wrapping and compaction, this project will open up new research directions towards understanding myelin formation, plasticity, and disease in the central nervous system.

NIH Research Projects · FY 2024 · 2020-09

PROJECT SUMMARY Professional burnout is a growing epidemic with symptoms affecting over 500,000 US physicians at any given time and carries significant adverse consequences for physician mental health, health care value, quality of care, and patient safety. There is an urgent need to develop reliable methods to proactively identify work environments at high risk for physician burnout, and tailor process improvements accordingly. The objective of this proposal is to develop and validate a real-time prediction model that uses operational data in primary care practices to identify high-risk clinics for physician burnout, enabling timely and tailored process improvements. The central hypothesis is that routinely-collected electronic health record (EHR) usage metrics coupled with practice-specific metrics can predict physicians’ risk for burnout and inform process improvement. The rationale for the proposed research is that early identification of high-risk clinics will allow organizations to tailor interventions to those clinics before burnout and its individual or health system consequences arise. This would be an innovative approach that prevents burnout rather than reacting to it. This project capitalizes on routinely-collect data from Stanford primary care clinics to create a database encompassing EHR usage metrics and practice-specific metrics. The specific aims are: Aim 1: Develop a prediction model to quantify risk for physician burnout. The working hypothesis is that real-time metrics of practice efficiency tracked by the EHR and other practice-specific metrics can predict physician burnout using a machine learning approach. Aim 2: Refine the prediction model using qualitative methods. The working hypothesis is that qualitative assessment will inform refinements to the prediction model created in Aim 1, and will demonstrate the face validity of the model. Aim 3: Validate the use of the prediction model to identify high-risk clinics. The working hypothesis is that quality of care metrics will demonstrate the concurrent validity, that subsequent routinely administered burnout surveys will demonstrate the predictive validity of the prediction model created in Aims 1 and 2, when aggregated at the clinic level. This proposal is significant because physician burnout is a growing problem with important implications for patient safety. It is also innovative in deploying machine learning and mixed methods to identify physicians at increased risk for burnout which will enable testing interventions to reverse this trend. In combination with formal training in quantitative and qualitative methods, expert mentorship, and participation in selected scholarly activities at Stanford, the experience gained through this project will facilitate progress toward a long-term goal to become an academic leader advancing evidence-based reform of the health care delivery system to optimize human factors that improve quality and safety.

NIH Research Projects · FY 2024 · 2020-09

SUMMARY The Stanford U54 SARS-CoV-2 Serological Sciences Center of Excellence (SUSS-COE) has remained true to its original goals as a member of the SeroNet consortium organized to address the urgent need for better understanding of human immune responses to the SARS-CoV-2 coronavirus in the Covid-19 pandemic. We have incorporated new facts into our research plans as they arose during the pandemic, including the availability of mRNA vaccines, the development of long Covid by a subset of infected individuals, and rare reactions towards vaccines. The preponderance of our work has been toward understanding the adaptive immune responses to SARS-CoV-2 and its variants in the context of infection or vaccination, with emphasis on responses occurring in non-blood tissue sites, and analyses of diverse populations. In the proposed Supplement funding for this award, we will carry out further investigations into SARS-CoV-2-specific responses in pregnancy; multi-antigen variant B cell sorting and single-cell transcriptome and B cell receptor sequencing experiments to deeply characterize the evolution of B cell memory through the pandemic; structural studies of protective antibodies bound to their antigens; extensive analysis of class I epitope presentation of SARS-CoV-2 across human HLA alleles; and in- depth T cell and B cell lineage analysis in patients with long Covid. The combined impact of these studies will be to extend understanding of the cellular and molecular mechanisms affecting immune responses to the continuing evolving SARS-CoV-2 viral populations in individuals with different prior exposure histories, and in some populations such as pregnant individuals or long Covid patients who have altered immunity.

NIH Research Projects · FY 2025 · 2020-09

Project Summary Abdominal aortic aneurysm (AAA) disease is a common cause of premature death in adult Americans. To date, no medical (e.g., non-surgical) therapies have proven effective at limiting AAA disease progression, or reducing the risk of AAA rupture or aneurysm-related sudden death. The recognition that diabetic individuals are less likely to develop AAAs and when present in diabetics, AAAs enlarge less rapidly and rupture less frequently, introduces new possibilities for medical AAA disease management. Recent retrospective studies suggest that metformin, the world’s most commonly prescribed oral hypoglycemic agent, may be associated with reduced rates of AAA enlargement. To date, however, the ability of metformin to suppress AAA disease has not been evaluated in a scientifically rigorous, prospective fashion. Building off existing observational evidence and novel preliminary data, generated to support this proposal, it is our fundamental hypothesis that metformin therapy will safely suppress AAA disease progression in non- diabetic patients. To test this hypothesis, two Specific Aims are proposed. The First Aim will evaluate the tolerability and safety of metformin in nondiabetic patients with AAA disease. Tolerance will be assessed by the serial administration of quality of life surveys and tracking participant compliance and retention. Safety will be assessed by semi-annual examinations, review of the source medical record, supplementary hematologic and metabolic panel surveys as needed. The Second Aim will test the ability of metformin XR (extended release) to reduce the average annual rate of enlargement of existing small to intermediate size AAAs by ≥ 30% compared to placebo. For this Aim, 480 participants will be randomized 1:1 to metformin or placebo. The primary endpoint will be the increase in mean maximal orthogonal AAA diameter through 24 months, as determined by computed tomographic aortography (CTA). Successful completion of these Aims will advance the understanding of AAA disease as well as the translational utility of metformin therapy to treat cardiovascular diseases in nondiabetic patients. These Aims specifically address the NIH Strategic Vision Goals of 1) understanding human biology, 2) reducing human disease, and 3) advancing translational research, as well as Objectives of 1) understanding normal biologic function and resilience, 2) investigating newly discovered pathobiological mechanisms, and 3) developing and optimizing novel therapeutic strategies to prevent, treat and cure HLBS diseases.

NIH Research Projects · FY 2025 · 2020-09

PROJECT SUMMARY/ABSTRACT The Stanford Clinical Center (SCC) submits a renewal application to continue participation in the Type 1 Diabetes in Acute Pancreatitis Consortium (T1DAPC). The SCC will continue to recruit and retain participants into the signature prospective observational cohort study – the Diabetes RElated to Acute Pancreatitis and its Mechanisms (DREAM) study. The SCC will recruit into the optional arms of the study including the mixed meal tolerance test arm, the frequently-sampled intravenous glucose tolerance test arm, and the MRI imaging arm. The SCC will continue to work collaboratively to pursue the comprehensive immunological analyses planned upon DREAM cohort completion. In anticipation that DREAM enrollment will be completed in the next grant period, the SCC has assembled a multi- disciplinary team including the disciplines of pancreatology, endocrinology, radiology, and immunology to develop and complete current and forthcoming secondary and ancillary study proposals approved by the T1DAPC Steering Committee.

NIH Research Projects · FY 2025 · 2020-09

In response to the NIH RFA-RM-19-009, we propose to establish the Stanford-SLAC CryoET Specimen Preparation Service Center (SCSC) to accomplish four specific aims: (1) establish a platform, which is available to the scientific community at large, to streamline the preparation of samples suitable for downstream cryogenic electron tomography (cryoET) data collection; (2) provide access to advanced cryo-specimen preparation techniques for a wide range of samples: macromolecular complexes, microcrystals of biological materials, cell lysates, organelles, cells and tissues; (3) generate a training curriculum for new users on the preparation of frozen, hydrated biological specimens, with the option of carrying out correlative cryo-fluorescence light microscopy (cryoFLM), cryo-focused ion beam scanning electron microscopy (cryoFIB), and cryoET; and (4) adopt new methods for sample preparation using innovative technologies developed elsewhere. In this proposal, we will leverage our existing facilities and in-house expertise in each of the following imaging modalities and their integration into a correlated sample preparation workflow: cryoFLM, cryoFIB, and cryoET. We will establish all the necessary equipment dedicated to the proposed service and training activities. We will prepare video curriculum materials to train new users in the aforementioned protocols. We will adapt our existing infrastructure to manage project administration and resource allocations. Administrative support is in place to help users with lodging arrangements, as well as laboratory on-boarding and specimen biosafety approvals. We have a well set- up communication infrastructure for remote users to participate in actual experiments, particularly for cryoFLM, cryoFIB, and cryoET. We will provide access to existing Talos Arctica and/or Titan Krios microscopes to evaluate whether the prepared samples are ready for data collection in the associated Hub, which will be set up at another institution with separate NIH support. We will make our existing Stanford-SLAC cryo-specimen preparation equipment available on a limited basis immediately after this proposal is funded. We anticipate serving ~40 users per year once the Center is fully operational in early Year 2 of the award period. We will also offer regular hands- on workshops to train ~10-12 new users per year. We will disseminate our resources to the broad community via a web portal and booths at professional societies’ annual conferences. The number of users will increase in subsequent years as we and our recurring users optimize the use of the Center’s resources. In Years 3-6, we will work closely with collaborators pioneering advanced protocols such as semi-automated and automated cryoFIB and “lift-out” for tissue samples to implement them in the proposed Center. Lastly, we will assemble a Scientific Advisory Committee of experts to guide the practices of our Service Center and the ongoing implementation of cutting-edge technologies.

NIH Research Projects · FY 2025 · 2020-09

Project Summary Despite the number of people affected each year by persistent pain after trauma or surgery the key components of the profound multicellular response to injury and how they can be manipulated to improve outcomes remains unclear. Peripheral injury mobilizes the immune system to resolve tissue damage, however, sustained immune activation can delay healing. Myeloid-lineage cells are instrumental in the innate immune response to injury- peripherally, as macrophages, and centrally, as yolk sac-derived microglia. Nevertheless, the time and compartment-specific contributions of myeloid cells to perioperative pain and successful recovery have yet to be elucidated. How to regulate this delicate balance, between pro-resolution and pro-inflammatory contributions of myeloid cells is a crucial topic our lab has been interested in for many years. During the initial grant cycle we have made several key discoveries in this area: 1) Different mouse models of peripheral injury exhibit distinct spatial and temporal accumulation of macrophages at the site of injury providing a potential biomarker for diagnosis and myeloid-targeted therapy monitoring, 2) Microglia exist in a range of heterogeneous states, including a “pro-resolution” state which contributes to recovery after injury and exhibits a unique transcriptome, 3) The intersection of mouse microglial sequencing data with a newly generated human spinal cord single microglial nuclei dataset using advanced bioinformatic analyses provides genes of interest for further study. These findings, and the technical capabilities we have developed during the prior cycle, uniquely position us to address the following key knowledge gaps over the next 5 years: 1) The myeloid immune response can be pro- inflammatory or pro-resolution, can these states be monitored in vivo to understand poor or successful recovery? 2) What specific molecular signatures of pro-resolution microglia can be targeted to improve outcomes? 3) Can we leverage microglial heterogeneity in humans to discover better translational targets? Precise manipulation of myeloid-lineage cells to establish causation is not possible in humans, however, the overall vision for the research program is to perform “clinically-informed basic science” by observing variables most significant to human recovery and testing them systematically in pre-clinical models. This will include consideration for sex and age as biological variables, the latter being particularly important as age is a major risk factor for injury and poor recovery. Our approach integrates concepts of injury mechanism (using varied mouse models), cell location (evaluating both peripheral and central involvement) and cellular heterogeneity (recognizing that myeloid cells exist in a range of states). Successful completion of the proposed studies will enhance our understanding of compartment-specific myeloid cell effects on healing after injury, identify cell- specific targets for intervention, and clarify when and in whom such treatments will provide the most benefit.

NIH Research Projects · FY 2024 · 2020-09

Project Summary/Abstract Increasing prevalence of Alzheimer’s dementia (AD) is a growing health and economic crisis. Although studied for 112+ years, the root causes for sporadic AD—which is > 95% of AD—are unclear. Over the last 15 years, 415+ clinical trials to test new drugs against AD failed. Approved drugs can only manage symptoms. I will use NIH Pioneer funding to investigate a novel hypothesis for the etiology of sporadic Alzheimer’s dementia, based on my insight that imbalance between two innate immune peptides may be a key factor that modulates the risk of formation, the stability, and clearance of AD-associated fibrils and plaques. Recent observations of chronic P. gingivalis and Herpesvirus infections being associated with Alzheimer’s fit this hypothesis. I am, to my knowledge, the only researcher working on this idea. The human cathelicidin LL-37, unique in our proteome, is an antiviral and antibacterial defense peptide deployed by microglia, macrophages, neutrophils, epithelia, B cells, and NK cells (to kill infected cells). Thus LL-37 is a centrally important defense peptide, necessary for killing bacterial and viral pathogens and infected host cells. LL-37’s Vitamin D3-, RXR-agonist-, and butyrate-dependent expression is also stimulated by infection, wounding, exercise, and some vaccines (e.g., BCG & OPV vaccines). Certain pathogens, P. gingivalis in particular, release enzymatic virulence factors that rapidly degrade LL-37. Degradation of LL-37 could well dysregulate the brain’s innate immunity, causing neurodegeneration; in LL-37’s absence, the immune process of macroautophagy is crippled. The Alzheimer’s-associated peptide Ab now seems also to be a host defense peptide; brain infections by either Herpesviridae or P. gingivalis stimulate Ab production, causing it to accumulate in plaques that co-locate with pathogens. Recently I and collaborators showed that LL-37 and Ab are both expressed in human brain, and bind each other sequence-specifically. LL-37/Ab binding prevents fibrillization and blocks Ab from adopting b-type secondary structure. Thus, LL-37 degradation may allow Ab to accumulate. Our in vivo studies show that cathelicidin induction in 5XFAD mice slows AD progression and improves 5XFAD cognition to match wild-type. I aim to tie this finding to infection-associated dementia. In this Pioneer project, I will use wild-type and cathelicidin KO mice to demonstrate that degradation of LL-37 by P. gingivalis virulence factors may well be one cause of brain tissue degradation leading to dementia, which can be prevented by early upregulation of cathelicidin to prevent infection; or treated orally with antimicrobials. My lab has developed new antimicrobials that potently kill both P. gingivalis and inactivate Herpesvirus.

NIH Research Projects · FY 2024 · 2020-09

PROJECT SUMMARY This R25 application entitled “The Stanford Pre-Renal Initiative: Undergraduate Training in Kidney Health” seeks to develop a thriving research training program for undergraduate students in the NIDDK and KUH mission areas of adult and pediatric nephrology and (benign) urology. Key features of the Program include: 1) an energetic, highly motivated Program Director with a track record of NIH-funded research, mentorship, and teaching; 2) an experienced, dedicated Advisory Council comprised of three Department Chairs and two Senior Associate Deans, including the Dean for Faculty Development and Diversity; 3) a talented, diverse Training Faculty from within and outside the host Divisions and Departments, with a broad array of research interests in basic, translational and clinical research; 4) a recruitment plan to host students from Stanford and seven regional undergraduate institutions with a commitment toward recruitment of women, underrepresented minorities, and undergraduates from other disadvantaged backgrounds; 5) well-established and time- tested methods of tracking progress of individual trainees; 6) a Nephrology and Urology training program with a truly exceptional level of focus and dedication to diversity recruitment; 7) an outstanding track record of training and mentoring in Nephrology and Urology with 582 publications or abstract presentations, 329 (57%) with trainees as lead author; and 8) didactic coursework and workshops with a focus on introducing Nephrology and Urology to potential, future kidney physicians and scientists. We propose to host eight students to participate in a 10-12-week summer research program accompanied by two didactic threads on: 1) research techniques and 2) application of life sciences to kidney physiology and workshops designed to prepare undergraduates for the next phase of their careers with inspiration to continue training in kidney health.

NIH Research Projects · FY 2024 · 2020-09

PROJECT ABSTRACT/SUMMARY Immunotherapies that enhance the ability of T cells to recognize and kill tumor cells have been transformational in the treatment of human cancer, but immunotherapy is not effective in all patients or cancers, and therefore studies interrogating the molecular basis for durable T cell responses to cancer are needed. A critical barrier for the sustained activation of tumor-infiltrating T cells is the development of T cell ‘exhaustion,’ which leads to the stable expression of inhibitory surface receptors, poor response to tumor antigens, and low cell proliferation and persistence of T cells in vivo. However, to date, it has been difficult to study the gene regulatory mechanisms that control the development of T cell exhaustion in humans, due to a lack of sensitive genomic tools to study primary immune cells from patients. We recently developed a suite of high-throughput epigenomic technologies that enable the measurement of three-dimensional (3D) genome conformation and single-cell chromatin accessibility in primary T cells from human tumors. In the proposed research, we aim to utilize these methods to identify changes in 4D nucleome (4DN) organization and accessibility that underlie the development of human T cell exhaustion. In Aim 1, we will define 3D genome interactions that occur in human T cell exhaustion in patients with advanced skin cancer. Exhaustion-associated genome conformation will be compared across several cancer types to identify a consensus exhaustion profile, and these findings will be integrated with chromatin accessibility and gene expression data to identify transcriptional effects of 3D changes. In Aim 2, we will determine the dynamics and reversibility of regulatory 3D interactions in exhaustion using a novel chimeric antigen-receptor (CAR)-T cell model. In Aim 3, we will perturb these interactions using CRISPR/Cas9 genome editing in primary T cells, coupled with single-cell epigenomic read-outs, to engineer improved, durable, next- generation immunotherapies. If successful, these findings will have a direct impact on the future design of immunotherapy strategies, which will have a significant impact on the clinical care of cancer patients. Finally, we will facilitate the dissemination of these findings by freely distributing protocols and data and releasing custom software tools, and we will use these studies as a collaborative launch point in the 4DN network. We anticipate that these results will lead to novel insights into the molecular regulation of T cell exhaustion and serve as an effective research program for Dr. Satpathy to establish his independent laboratory at the interface of immunology and genome science.