Fred Hutchinson Cancer Center

NIH Research Projects · FY 2025 · 2024-08

PROJECT SUMMARY Adoptive transfer of engineered T cells targeting tumor antigens is ineffective against most solid tumors, due in-part to poor persistence and the rapid onset of dysfunction. Recent studies of endogenous tumor- specific T cells (Endog-T) have described a population of stem-like PD-1+Tcf1+ progenitor exhausted T cells (Tpex) that can self-renew and differentiate into effector-like T cells in the tumor. Tpex preferentially reside in tumor-draining lymph nodes (TdLNs), where they are maintained through activation by dendritic cells (DCs). Importantly, Tpex in TdLNs are necessary for promoting persistent and functional T cell responses, as well as conferring response to PD-L1 blockade. However, it is not clear whether engineered T cells form PD-1+Tcf1+ populations in TdLNs, and how this impacts their long-term persistence and function in the tumor. To test this, I adapted the clinically relevant KrasG12D-LSL/+;p53fl/fl murine model (KP) of lung adenocarcinoma to overexpress the model neoantigen Ovalbumin (Ova) to compare Ova-specific engineered T cells (TCR-T) and Endog-T cells. While the majority of Endog-T cells in TdLNs were PD-1+Tcf1+, TCR-T cells were predominantly PD-1-Tcf1+, markers associated with lack of activation and maintenance of a pre-exhausted central-memory phenotype. Consistent with these phenotypes, PD-L1 blockade failed to increase numbers of TCR-T cells and enhance tumor control in comparison to Endog-T. Thus, I hypothesize that factors unique to TCR-T therapy, such as their in vitro pre-activation and/or infusion at a high cell number, promote their formation of PD-1-Tcf1+ reservoirs in TdLNs instead of PD-1+Tcf1+. I also hypothesize that formation of PD-1- Tcf1+ reservoirs in the TdLN is actually an advantage for TCR-T therapy, since the PD-1-Tcf1+ population may possess superior functional potential that can be mobilized through vaccination targeting DCs in the TdLN. In this project, I seek to determine 1) why TCR-T cells form PD-1-Tcf1+ reservoirs in TdLNs instead of PD- 1+Tcf1+ like what is described for Endog-T, as well as 2) whether the PD-1-Tcf1+ TCR-T cells in the TdLN have stem-like characteristics and functional potential that can be unleashed with vaccination. In Aim 1, I will use fluorescently-labeled tumors in the KP model, flow cytometry, and high-parameter fluorescence microscopy to determine how infusion of a large number of cells and/or in vitro pre-activation affect TCR-T cell formation of PD-1+Tcf1+ populations in TdLNs, and how this correlates with their localization near DCs. In Aim 2, I will use RNA-seq, ATAC-seq, and ex vivo assays to describe the stem-ness and functional potential of the PD-1-Tcf1+ TCR-T cells in the TdLN compared to PD-1+Tcf1+ Endog-T. I will also evaluate whether a novel T cell vaccination approach targeting DCs in TdLNs can activate PD-1-Tcf1+ TCR-T cells to promote tumor control. Through this work, I will define the mechanisms by which TCR-T cells form Tcf1+ stem-like reservoirs in TdLNs and potentially identify a therapeutic strategy to enhance TCR-T efficacy by targeting these reservoirs that could be translated to the clinic to improve patient outcomes.

NIH Research Projects · FY 2025 · 2024-08

PROJECT ABSTRACT/SUMMARY Alzheimer's Disease and related dementias (AD/ADRD) are common and debilitating conditions. Financial hardship, a multidimensional construct of financial strain, financial stress and asset depletion, is common in AD/ADRD due to exorbitant out-of-pocket spending such as for long-term care, lower work productivity and income for their caregivers that can last for decades after disease onset, and difficulty deciding between nursing home care or home-based care while negotiating insurance coverage. People from historically marginalized groups can experience a double disparity with fewer financial resources to manage AD/ADRD and a greater risk of AD/ADRD. Screening for financial hardship in AD/ADRD is key for addressing the needs of patients and caregivers but critical barriers include a lack of suitable screening measures. Current measures are very general and meant for people without chronic medical conditions or are specific to other diseases. To fill this gap, this study will create a suite of measures that can screen for financial hardship in people with AD/ADRD and their families and caregivers. The measures will include a set to assess caregiver burden; a set to assess patient hardship as reported by the caregiver for patients who cannot report for themselves; and a set of patient-reported measures for patients that are able to report for themselves. To create these financial hardship screening measures, the project will conduct the following aims. Aim 1- Develop financial hardship screening measures for Alzheimer's Disease and related dementias: Using interviews with both caregivers and people with AD/ADRD, key indicators of financial hardship that are unique to AD/ADRD and the point in the lifespan in which it occurs will be identified. The ways that social and caregiver network size affect financial hardship will also be explored. Using the interviews and previous measures, preliminary measures will be created and will be reviewed by experts and a patient and caregiver advisory board. Aim 2- Create item response theory-based screening measures for financial hardship measures in Alzheimer's Disease and related dementias: Large samples of people with AD/ADRD (n=1000) and caregivers (n=1000) will be surveyed and item response theory will be used to evaluate and revise the measures and create scoring algorithms. A sample of additional caregivers matched to primary caregivers (n=400) will also be recruited to evaluate interrater reliability of the measures. Aim 3- Evaluate the financial hardship measures across patient and caregiver populations: Using the sample from Aim 2 and item response theory, we will evaluate the financial hardship screening measures across the following groups to ensure they are unbiased and reflect true differences: race/ethnicity; patient comorbidities; stage of AD/ADRD; caregiver relationship; social network size; number of caregivers; financial support provided; and caregiver's own health status (disability, comorbidities). The resulting measures will improve identification of financial hardship in AD/ADRD.

NIH Research Projects · FY 2024 · 2024-08

PROJECT SUMMARY/ABSTRACT The incidence of hematological malignancies is higher in persons living with PLWH (Persons living with HIV (Human Immunodeficiency Virus)). The risk of developing classic Hodgkin lymphoma (CHL) in PLWH is 5-26 times the general population. The US (United States) HIV/AIDS Cancer Match study documented a 1.63-fold increase in the incidence of CHL in PLWH after the introduction of ART compared to the pre-ART era. CHL is an unusual B-cell malignancy with <5% tumor cells amidst >95% benign cells composed of immune and stromal cells. Most are of the mixed cellularity histological subtype of CHL, and 80-100% of CHL in PLWH are EBV-associated. Our previous study, using a limited panel of antibodies, showed that the immune microenvironment of CHL in PLWH differs from those seen in immunocompetent patients. Furthermore, the immune microenvironment plays a crucial role in the pathogenesis of CHL. DNA from leukocytes in untreated HIV-infected individuals show DNA methylation changes indicative of aging. Combination ART can partly reverse epigenetic aging but does not entirely correct it. We hypothesize that the persistent epigenetic alterations within the immune cells in PLWH on long- term combination ART result in altered and aberrant gene expression in the immune cells. In the context of the presence of sufficient numbers of CD4+ T cells, the abnormal immune cells contribute to the pathogenesis of classic Hodgkin lymphoma; they are responsible for the increased relative risk of developing CHL in PLWH on combination ART. We propose to identify persistent transcriptomic changes in immune cells in benign lymph nodes of PLWH on combination ART and address whether these or related changes can be documented in CHL samples of PLWH. We will select benign/reactive lymph nodes from 21 PLWH on ART and 21 age-matched non-PLWH groups, and CHL samples from 26 PLWH and 26 age-matched and EBV-association matched non-PLWH individuals. We will include paired benign lymph node and CHL samples from the same patients in the study. RNA extracted from the samples will be evaluated by Bulk RNA Barcoding and Sequencing (BRB-seq) and by Nanostring nCounter analysis. For nCounter analysis, we will use a panel ~100 genes representative of immune response to hematolymphoid tumors, to identify differentially expressed RNAs and to validate the results of BRB-Seq. Tissue microarrays will be prepared from the paraffin blocks, and spatial transcriptomics and protein expression will be undertaken using the CosMXTM Spatial Molecular Image analysis system to evaluate the tissue microenvironment in a single-cell and spatial context. The differential RNA expression not addressed by CosMXTM will be addressed by multiplex-immunohistochemistry and in-situ hybridization (RNAscope). The innovative BRB-seq that we propose to validate will significantly reduce the costs of RNA sequencing and expression analysis.

NIH Research Projects · FY 2025 · 2024-08

Abstract Novel immune therapies are needed to help the majority of patients with melanoma and other solid tumors who fail to benefit or stop responding to immune checkpoint inhibitors, and adoptive transfer of tumor specific T cells is one method that con overcome resistance to other immune therapies. Most research has focused on engineering CD8 T cells that directly target and kill cancer cells, but this proposal will focus on the “helper” CD4 T cells that work by orchestrating the function of other immune cells. The goal of this proposal is to develop a therapeutic strategy using CD4 T cells to overcome treatment resistance in solid tumors. Correlative studies from patients with cancer show that having greater numbers of tumor specific CD4 T cells are associated with activation of other immune cells in tumors and better outcomes. Work in mouse cancer models show that CD4 T cells can work through support of CD8 T cells and through innate immune cell killing of tumors even when other T cells are not present. The most effective CD4 therapies in the mouse cancer models have combined CD4 T cells with other signals that activate innate immune cells in tumors, but these signals are toxic when given throughout the whole body. We have found that two potent innate immune signals, IL-12 and IL-18 can be engineered into CD4 T cells that can target these signals specifically to tumors. We find that CD4 T cells engineered in this way can destroy tumors, activate CD8 T cells, and activate immune cells in mouse cancer models. In our first aim we will identify which molecules made by T cells are important in this process, which immune cells in tumors are the targets, and which immune cells are responsible for tumor cell death. Use of this approach in patients with melanoma will require ways to produce large populations of CD4 T cells that are specific to the patients’ cancer, and we will obtain these cells from surgically removed tumors from patients. Existing methods to grow tumor specific T cells from tumors have had some benefit, but the CD4 T cells are mostly lost during the standard process. We have identified a way to identify the tumor specific CD4 T cells within a tumor and will use this information to isolate these cells away from other T cells so that we can grow them. In our second aim we will develop a reliable process to isolate and expand these cells that maintains their function, as well as engineer them with human versions of the IL-12 and IL-18 signals we explored in mice. We will then use a humanized mouse model to test whether CD4 T cells grown from human tumors can effectively target human tumor and immune cells. The goal of this work is to develop a new type of treatment for cancer using CD4 T cells engineered with innate immune signals that can be used in clinical trials of patients with treatment resistant melanoma and other solid tumors.

NIH Research Projects · FY 2025 · 2024-08

Project summary/abstract Human immunodeficiency virus infects cells of the immune system and, if left untreated leads to the development of acquired immunodeficiency syndrome and death. With the advent of antiretrovirals, HIV infection has turned into a chronic infection. However, residual viral transcription is associated with increased risk for co-morbidities in people with HIV infection. Programmed cell death protein 1 (PD-1) is an immune checkpoint protein expressed on latently infected CD4 T cells and in particular T follicular helper (TFH) cells. TFH cells are located in the germinal centers and are enriched in replication-competent human immune deficiency virus 1 (HIV) provirus in people with HIV. My proposal builds upon the pre-clinical work I performed in non-human primates in which we demonstrated that anti-PD1 chimeric antigen receptor T cells eliminated all detectable PD-1 expressing TFH cells; the first time the elimination of this reservoir of HIV has been reported. Unfortunately, it also depleted memory CD8+ T cells substantially, which led to accelerated disease progression. Provided we engineer higher safety and specificity, the high potency of anti-PD1 CAR T cells suggest it offers promise for a functional HIV cure. The goal of the proposed project is to define the genetic architecture of a safer and more specific second-generation anti-PD-1 CAR. Aim 1 is divided in two sub aims that describe the development of first, a more specific anti-PD- 1 CAR by integrating its expression under the control of an additional TFH-specific synNotch receptor and second, a safer CAR by adding an ON switch controlled by a small molecule inhibitor. These experiments will demonstrate that a second-generation anti-PD-1 CAR is highly specific in depleting TFH cells and has the potential to abrogate viral replication within B cell follicles more specifically, thereby providing foundational knowledge to enable further studies towards an HIV cure. My career goals are to become an assistant professor at a top-tier academic research institution. I aim to lead a research program that investigates cell therapies that can contribute to end the HIV epidemic by providing a functional cure on an individual level, thereby decreasing the number of HIV carriers over time. To this aim, I will receive training to use a NHP lentiviral infection model in to develop next-generation HIV cure therapies, while expanding my knowledge of cell engineering. Dr. Lawrence Corey (FHCC) will act as the primary mentor, who has substantial experience in HIV persistence, development of antiviral agents and clinical trials, while supported by an Advisory Committee with expertise in gene and cell therapies, and NHP models of HIV. I will present my work at international conferences and to my mentoring committee. I will receive training in laboratory management/leadership, grant writing, negotiation, and mentoring to help me lead a successful research team. FHCC has superb research facilities, professional development resources and administrative support, which will provide the infrastructure to support my research and career development as a mentored staff scientist and ultimately, my transition to independence.

NIH Research Projects · FY 2025 · 2024-08

Project Summary Chronic Hepatitis B virus (HBV) infection (CHB) affects hundreds of millions of people across the world. Despite effective vaccines, and antiviral therapies that can suppress viral replication, these infections are largely incurable, and new therapies for CHB are desperately needed. We have investigated gene editing using targeted nucleases to cleave covalently closed circular DNA (cccDNA), the template for HBV replication, which results in its degradation or inactivation in infected hepatocytes. Our work and the work of others has been hindered due to the fact that existing small animal models for HBV have substantial limitations, or are complex and expensive to establish. To this end, we established and validated a robust and low-cost liver humanized NSG-PiZ mouse as a model for the study of HBV. Here we propose to use these mice for the evaluation of novel curative gene editing therapies that target CHB. Specific Aim 1. Evaluate the anti-HBV efficacy of HBV gene therapy in liver-humanized NSG-PiZ mice. We will first evaluate the efficiency of HBV gene editing in NSG-PiZ mice, using AAV-SaCas9 vectors we previously validated in HBV-infected humanized FRG mice. We will then compare lipid nanoparticle (LNP) delivery of nuclease mRNA with AAV vectors for the transduction of PHH in our model. We will compare the antiviral efficacy of different classes of gene editing nuclease in our NSG-PiZ mouse model of CHB, and evaluate chromatin modifying agents for their ability to make cccDNA accessible to gene editing nucleases in vitro and in vivo. Finally, we will assess combined gene-editing, reverse transcriptase inhibitor (RTi), and entry inhibitor antiviral therapy in our model of CHB. Specific Aim 2. Evaluate the tolerability and host genomic consequences of AAV- and LNP-delivered nuclease therapies targeting HBV in liver-humanized NSG-PiZ mice. We will first determine the maximum tolerated dose (MTD) and minimal effective dose for AAV-nuclease and LNP-nuclease therapies in our CHB model. We will then monitor the effects of different nuclease classes on treatment-related off-target genotoxicity in vivo and determine whether use of liver-specific promoters can mitigate therapy-associated genotoxicity. We will also determine whether therapy-associated genotoxicity is reduced when nuclease expression is transiently provided by LNPs. Finally, we will evaluate therapy-specific immune responses and therapy- associated hepatotoxicity and neurotoxicity after transient or persistent nuclease expression in immunocompetent mice.

NIH Research Projects · FY 2025 · 2024-08

PROJECT SUMMARY Kaposi sarcoma-associated herpesvirus (KSHV) is an oncogenic virus that causes Kaposi sarcoma (KS), one of the most common malignancies in people living with HIV worldwide. KSHV also causes primary effusion lymphoma (PEL) and multicentric Castleman disease (KSHV-MCD). Infection is common in sub-Saharan Africa and often occurs early in childhood, while in the western hemisphere, infection is rare in the general population but more frequent in men who have sex with men. A vaccine that prevents KSHV infection and/or associated morbidity and mortality represents a critical unmet need. However, the types of immune responses a vaccine would need to elicit have not been well defined. Here we will seek to identify the relevant antigenic targets and specific epitopes targeted by KSHV-neutralizing antibodies and to establish the ability of neutralizing antibodies to protect against KSHV infection in a small animal model. To do this, we will leverage a large pre-collected repository of serum and PBMC samples from KSHV+ participants with and without KS disease to characterize the neutralizing antibody response to KSHV. We will produce recombinant KSHV glycoproteins that will be used to deplete serum binding antibodies and assess their contribution to the polyclonal serum neutralizing response. In parallel, we will isolate neutralizing monoclonal antibodies from KSHV+ PBMC samples and carry out in-depth structure-function analyses to identify critical sites of vulnerability on KSHV glycoproteins. Lastly, we will evaluate the ability of neutralizing KSHV monoclonal antibodies to prevent infection in a humanized mouse model of KSHV infection. Our studies will define the relevant antigens and epitopes targeted by neutralizing antibodies that arise from natural KSHV infection and thus inform the design and development of KSHV vaccines. Moreover in vivo protection from KSHV infection will establish critical proof of concept that a KSHV vaccine should seek to elicit neutralizing antibodies.

NIH Research Projects · FY 2025 · 2024-08

PROJECT SUMMARY/ABSTRACT While immunotherapy is transforming cancer treatment, the majority of patients do not achieve durable responses. We have been studying response and resistance to different immune checkpoint inhibitors and are now poised to propose mechanistic studies aimed at providing an understanding of the immune states and pathways that mediate or inhibit response to immune checkpoint blockade. Using high-dimensional unbiased single-cell RNA-seq (scRNAseq), we can identify both canonical and non-canonical immune effectors that can mediate anti-tumor responses. We believe that non-canonical effectors such as cytotoxic CD4 T cells, which we have recently described, are not effectively triggered by our current treatments. Using the same single-cell approaches, we can identify both known and novel cell types in cancer patients that can mediate immune suppression. In our first objective, we will determine whether combination immunotherapies that include drug(s) targeting specific immunosuppressive cells can enhance the function of these cytotoxic CD4+ T cells. By leveraging neoadjuvant clinical trials where patients receive immunotherapy prior to surgery, we will use single cell genomics and proteomics to define whether these combinations can 1) target the desired immunosuppressive mechanisms, and 2) enhance canonical and/or non-canonical effectors within the resected tumors. We will also use this approach to determine whether we can map these specific cell states into the circulating compartment. The second objective is based on a longstanding interest in our group to define the dynamics of antigen-specific responses. Using single-cell T cell receptor sequencing, we can identify expanded T cell clones as well as follow their localization. In addition to understanding how immunotherapy combinations induce and modulate specific T cell clonotypes within the tumor, we can determine how immunotherapies can induce functional plasticity to desired or undesired states. The third objective builds on our 20 year experience using mouse models to dissect mechanisms underlying response or resistance to immunotherapy. We will determine the functional significance of non-canonical immune effectors using depletion and knock-out strategies. We will also determine how combination immunotherapies can elicit both effective or ineffective anti- tumor immune responses, thereby guiding the design of future clinical trials. In conclusion, our proposal is based on hypothesis-driven bench-to-bedside and bedside-to-bench mechanistic studies with the goal of advancing cancer immunotherapy. With our deep expertise in this field, experience leading multi-disciplinary teams focused on translational research, and a rich network of basic science and clinical collaborators; we are uniquely positioned to succeed in the research plan outlined in this proposal.

NIH Research Projects · FY 2026 · 2024-08

PROJECT SUMMARY Uterine fibroids are the most common pelvic tumors in women of reproductive age, coming to clinical attention in one in four women. Although these smooth muscle tumors are non-malignant, fibroids are a significant medical problem, coming to clinical attention in one on four women and accounting for over 200,000 hysterectomies annually. Furthermore, fibroids consume a significant amount of health care resources, accounting for billions of dollars annually. Of note, fibroids are 2-3 times as common among Black women compared to White women. Despite significant quality of life and health care costs associated with fibroids, few modifiable, factors have been identified, nor is it understood why Black women are disproportionately affected. A major barrier to understanding the relative contributions of established and yet-to-be identified risk factors that may contribute to the fibroid difference is that no single study has adequate numbers of both Black and White women to apply advanced epidemiologic methods (e.g., mediation analyses, population attribute risks) to evaluate these differences. We propose to study women from two prospective U.S. cohorts — Black women from the Black Women’s Health Study (BWHS) and White women from the Nurses’ Health Study II (NHSII) — to identify factors that explain the differences in fibroid incidence between Black and White women. Among 59,000 participants (6,200+ newly diagnosed cases) in the BWHS and 116,429 (8,100+ newly diagnosed cases) in the NHSII, we will examine the following specific aims: Aim 1. Determine if established fibroid risk factors (e.g., parity, age at menarche, smoking, body size), which differ in prevalence or timing between Black and White women, contribute to racial differences in the risk of uterine fibroids. We will calculate race-specific population attributable fractions to identify the proportion of fibroids cases that are attributable to each risk factor and summarize the impact of differences in the distribution of fibroids risk factors by race on fibroids incidence using a novel average odds ratio method. Aim 2. Determine if environmental and neighborhood-level factors contribute to the racial differences in the risk of uterine fibroids. Aim 3. Estimate the contribution of multiple factors simultaneously to the fibroids incidence difference between Black and White women using multiple mediation analysis. Factors to be considered for inclusion into the mediation models include reproductive, lifestyle, psychosocial, and environmental factors. Given that more than 25% of all women experience fibroids, the potential health and economic impact of this study is substantial. Black women suffer disproportionately from fibroids, but the reasons for this difference are not understood. The identification factors that contribute to the racial difference in fibroid incidence could lay the groundwork for identifying strategies that would reduce fibroid incidence among all women.

NIH Research Projects · FY 2025 · 2024-08

SUMMARY/ABSTRACT HIV-1 remains a major public health challenge worldwide, with 1.3 million new infections and over 600,000 AIDS- related deaths in 2022. An effective vaccine that confers long-term protection against infection would be an essential tool in eradicating AIDS, but all vaccine candidates have been largely unsuccessful to date. This is due to the unique biology of the virus and its extraordinary ability to escape immune responses. The immune events immediately following HIV-1 transmission have critical impacts on the ensuing disease course, but the underlying biology has proven difficult to detail. Overall, the innate immune response to a viral infection can establish a state of viral resistance and contribute to controlling virus replication, but it also induces inflammation that can favor viral spread, immunopathology and disease progression. Studies of prospective cohorts of volunteers at high risk of contracting HIV-1 have revealed the sequence of events during acute infection, including the cytokine storm induced by the innate immune responses. Mechanistic in vitro studies have demonstrated the capacity of diverse innate immune sensors to detect HIV-1 and induce cytokine production. However, the relative contribution of each innate immune cell type and sensing pathway following in vivo HIV-1 infection, and their functional impact on both the virus and the host, are largely unknown. To overcome this limitation, we developed the MISTRG model of mice repopulated with a complete human immune system (“humanized mice”), including monocytes, macrophages, dendritic cells and NK cells that are essential to mounting innate immune responses. MISTRG mice are permissive for HIV-1 infection and recapitulate several aspects of the immune responses observed in humans, including the cytokine storm triggered in the first few weeks after infection. Furthermore, we have recently developed a highly efficient protocol to knockout genes from the human immune system of MISTRG mice, providing genetic tools that were previously exclusive to conventional mouse models. Using the innovative MISTRG model, we now plan to investigate the fundamental in vivo mechanisms of the innate immune response to HIV-1, following infection through both intravenous and mucosal routes. First, we will identify the cellular source of cytokines that are released during the acute phase of HIV-1 infection. We will complement these descriptive studies with functional depletion experiments, to determine how each cytokine-producing cell type affects the virus and the infected host. Second, we will use gene knockout approaches to determine the functional roles of membrane-bound versus intracellular innate immune sensors in the response to HIV-1. We hypothesize that different cell types contribute to HIV-1 sensing in vivo, that they rely on distinct sensors to do so, and that their concerted responses functionally impact the course of disease. Our findings will address a significant knowledge gap, and may inform the rationale design of novel innate immune interventions for the prevention or therapy of HIV-1/AIDS.

NIH Research Projects · FY 2025 · 2024-08

ABSTRACT HIV strains vary considerably in both genetic and phenotypic features, but all variants use CD4 as the primary receptor and consequentially CD4+ T cells are the major target for viral infection. However, variants may use two distinct co-receptors, CCR5 and/or CXCR4 to facilitate steps in the entry process subsequent to CD4 binding. Both CCR5-using viruses (R5 viruses) and CXCR4-using viruses (X4 viruses) are often present during chronic and later stages of HIV infection, but new infections are almost always seeded by R5 viruses. The host factors that contribute to this transmission bottleneck may hold clues to host biological determinants of HIV risk, but these factors have remained elusive. We have identified a host gene expressed in CD4+ T cells that restricts X4 virus replication and enhances R5 virus replication. Thus, this gene, SLC35A2, regulates HIV infection in a manner that mirrors what is observed during transmission. This is the first example of a gene that specifically restricts X4 virus infection. Because this gene was only recently discovered, its role in infection in mucosal CD4+ T cells, which are particularly important in sexual HIV transmission, has not been defined. We propose here to examine the effect of SLC35A2 on R5 versus X4 virus infection in primary mucosal CD4+ T cells using both single virus infections as well as virus competition experiments. Our proposed experiments will also test the hypothesis that SLC35A2, which is a transporter of UDP-galactose and thus plays a role in glycosylation, acts through the coreceptor early in the viral lifecycle, during fusion. These proposed high-risk, high-pay off studies have the potential to reveal a unique role for SLC35A2 in HIV transmission biology and address a long-standing puzzle in the field - namely why are R5 viruses selected for transmission.

NIH Research Projects · FY 2025 · 2024-07

PROJECT SUMMARY Endometriosis is a common and debilitating gynecologic disorder characterized by the presence of endometrial-like tissue outside of the uterus. It burdens approximately 10% of persons born with internal reproductive organs in the U.S., and incurs significant health care costs and morbidity. Prior research suggests that Black women are less likely to be diagnosed with endometriosis than White women; however, these racial/ethnic differences in endometriosis diagnosis likely reflect disparities in access to care and clinical presentation as opposed to biological differences in endometriosis incidence. To date, there has been almost no research on risk factors for or consequences of endometriosis among Black women. This is a significant gap in the literature as many established risk factors for endometriosis differ in their prevalence between Black and White women (e.g., age at menarche, parity, body mass index). Further, endometriosis is associated with higher risk of several diseases that disproportionately impact Black women (e.g., systemic lupus erythematosus). In recent work, patterns of the association between endometriosis and ovarian cancer risk differed between Black and White women, as did modifiers of the endometriosis-ovarian cancer association. These differences highlight the critical need for research into the impact of endometriosis on Black women across the life course. To address these important knowledge gaps we will utilize the Black Women’s Health Study, a prospective cohort of Black women followed to date for 27 years. This cohort represents a unique opportunity to evaluate the association between endometriosis risk factors and comorbidities among 59,000 participants, among whom over 1,600 endometriosis cases have occurred. Here we propose to further our understanding of endometriosis among Black women through the following specific aims: Aim 1. Determine whether existing risk factors that have been strongly and consistently associated with endometriosis in studies of predominantly White populations (i.e., parity, age at menarche, menstrual cycle length, body size, birth weight), are associated with endometriosis risk among Black women. Aim 2. Investigate the association between endometriosis and autoimmune diseases that have previously been associated with endometriosis and have a higher prevalence in Black women, including examining whether these associations vary by hysterectomy status. This proposed study is a unique opportunity for the first ever, large-scale prospective study on risk factors and consequences of endometriosis among Black women. The epidemiologic methods needed to examine endometriosis in a non-clinical setting are complex and the outcomes of this project will lay the groundwork for a larger scale grant where more novel risk factors and endometriosis consequences can be examined allowing for endometriosis research to expand beyond White populations.

NIH Research Projects · FY 2025 · 2024-07

PROJECT SUMMARY/ABSTRACT Keratinocytes, the major building block of skin epidermis, are the primary target of herpes simplex virus (HSV) infection as well as the mediator of local environmental cues, including those directed by CD8 tissue resident memory T cells (TRM) at the dermal epidermal junction (DEJ). The ability of keratinocytes to relay and amplify TRM immune signals could dictate the epithelium resistance or susceptibility to HSV infection and ultimately affect the outcome of HSV manifestation. Genital HSV-2 is a disease of global importance. There are an estimated 4 million incident HSV-2 infections yearly, with 572,000 in the US. HSV-2 is the underlying attributable risk in 30% of the incident 1.5 million HIV infections yearly. HSV-2 disease varies widely among individuals, ranging from disease free to infrequent and short-lasting recurrences to frequent and severe ulcerations. Subclinical shedding occurs commonly and frequently. The molecular mechanisms underpinning varied host responses to HSV-2 reactivation in genital skin are poorly defined. Understanding host-viral interactions, with the goal of enhancing responses to rapidly eliminate viral infection at the site of HSV-2 release before local viral dissemination, is the key to developing novel interventions and a successful vaccine. We have shown that CD8 TRM constitute the frontline of defense at the barrier surface. Our recent studies indicate CD8 TRM at the DEJ communicate with surrounding keratinocytes via IFN-γ to enhance their cell- intrinsic and innate antiviral responses through IFN-stimulated gene pathways. These observations alter the canonical belief that communication only goes from innate to adaptive immunity and suggest the reverse also occurs. This proposal is designed to dissect the cross talk between TRM and the surrounding epithelium and the microenvironmental signals that dictate the recruitment and function of CD8 TRM using sequential genital skin biopsy tissue in humans. By applying state-of-the-art single-cell RNA sequencing, spatial transcriptome analysis and highly multiplexed imaging technology, we aim to identify cellular and immunological characteristics that distinguish between effective vs defective antiviral responses and can be targeted for modification and manipulation to enhance immunity at the peripheral tissue. Findings generated in this study will provide rationale for eliciting optimal local immune responses and for development of a functional cure for HSV-2 recurrent disease in humans.

NIH Research Projects · FY 2025 · 2024-07

Project Summary/Abstract Tumor metastasis is responsible for the vast majority of deaths from epithelial cancers, including breast cancer. Understanding how cancer cells spread to distant sites, persist as dormant disseminated tumor cells (DTCs), and eventually recur is essential to improving the treatment of this disease. The long-term goal of my research group is to identify pathways that regulate these processes to enable the development of therapies that can prevent or treat metastatic recurrences. We recently identified a role for the antioxidant stress response transcription factor NRF2 in promoting the survival and local recurrence of mammary tumors following targeted therapy. We found that NRF2 is activated following diverse targeted therapies as a consequence of metabolic and oxidative stress. NRF2 remains constitutively activated in recurrent tumors, where it functions to promote recurrence through regulating redox homeostasis and nucleotide metabolism. While these results identify a function for NRF2 in local recurrence, a role for NRF2 in promoting metastatic dissemination remains relatively unexplored. Emerging evidence suggests that metastatic dissemination is associated with metabolic and oxidative stress. Consistent with this, we recently found that the NRF2 transcriptional program is elevated in metastatic tumors from patients with breast cancer. However, a thorough understanding of NRF2 regulation during metastatic dissemination, and how NRF2 activation functionally affects metastasis, remains unknown. This proposal will build on this work to define the regulation and function of NRF2 during metastasis in breast cancer. In new preliminary data, we found that NRF2 suppresses pro-inflammatory signaling in breast cancer. Consistent with this, NRF2 knockdown leads to infiltration of immune cells into the tumor microenvironment. Based upon these findings, the central hypothesis for this proposal is that NRF2 promotes breast cancer progression by inhibiting pro-inflammatory signaling between cancer cells and the tumor immune microenvironment. We will test this hypothesis through three specific aims. In Aim 1, we will define the mechanism and function of NRF2 regulation of the tumor immune microenvironment. In Aim 2, we will determine the role of NRF2 suppression of inflammatory signaling in promoting the outgrowth of disseminated tumor cells. In Aim 3 we will test whether targeting metabolic pathways can prevent the growth of NRF2-high tumors. Our work will reveal new information on how NRF2 functions to regulate tumor progression and may uncover novel therapeutic opportunities to prevent metastatic recurrence.

NIH Research Projects · FY 2025 · 2024-07

PROJECT SUMMARY (from parent grant) Pancreatic ductal adenocarcinoma (PDA) is a leading cause of cancer-related mortality in the United States. Unfortunately, both genomic and transcriptomic analyses of PDA have failed to identify therapeutically relevant targets. Combination chemotherapy remains the standard of care for the majority of patients who present with locally advanced or metastatic disease, resulting in a median overall survival of less than one year. PDA has been subclassified into 2-4 transcriptional subsets, which can be defined by their unique epigenetic states rather than a specific genetic profile. Of these subsets, the quasi-mesenchymal (QM) subtype is characterized by the worst prognosis, thus highlighting the importance of identifying new therapeutic avenues for QM PDA. The short- term goals of this proposal are to elucidate the genetic or epigenetic mechanisms regulating QM PDA subtype determination and to leverage that understanding toward the development of targeted therapies for QM disease. Our preliminary data suggest PDA may downregulate a novel epigenetic regulator and tumor suppressor in PDA in order to achieve this more aggressive QM PDA phenotype, while at the same time rendering tumor cells more sensitive to specific targetable therapies. This epigenetic regulator, a histone deacetylase called sirtuin 6 (SIRT6), was recently shown to act as a potent tumor suppressor in genetically engineered mouse models (GEMMs) of PDA, and inversely correlates with poor prognosis in patient samples. However, how SIRT6 is downregulated in PDA and its role in regulating PDA subtypes remains unknown. Here we propose three specific aims: (1) To identify novel mechanisms of SIRT6 downregulation in PDA; (2) To determine the functional role of SIRT6 in regulating PDA subtypes; and (3) To develop novel therapeutic approaches for this QM subset of PDA tumors. To accomplish these aims, we will apply a combination of cytogenetics, high- throughput sequencing, genetic gain of function and loss of function approaches and pharmaceutical interventions to a robust panel of molecularly characterized PDA GEMMs, human PDA cell lines and patient- derived xenografts from the NCI Patient-Derived Models Repository. Our approach also takes advantage of an innovative class of targeted therapeutics, which bind covalently to a specific residue on their target protein in order to achieve greater inhibition and specificity. The long-term goal of our work is to transform the clinical paradigm for this cancer from combination chemotherapy, toward a precision medicine-based approach utilizing predictive biomarkers to tailor more effective and less toxic therapies for PDA patients.

NIH Research Projects · FY 2025 · 2024-07

Project Summary/Abstract Demographic changes in the commercially insured population following implementation of the Affordable Care Act (ACA)—specifically improved enrollment of the near-poor and those with chronic comorbidities--may have paradoxically magnified the problem of “underinsurance;” that is, insurance that leaves persons at high risk for financial hardship and care gaps following illness. The central objective for this study is to understand the extent to which commercially insured cancer patients are at risk for shortfalls in care and adverse financial outcomes. Our working hypothesis is that underinsurance is causing gaps in care and financial hardship for a substantial proportion of cancer patients with commercial insurance in the post-ACA era. To address our objective and hypothesis, we will utilize a novel person-linked, population-level database that includes: 1) Western WA SEER cancer registry data for cancer patients diagnosed in 2009-2022; 2) WA state voter registration file data (non-cancer controls); 3) Enrollment and claims records from the state’s largest commercial payers (Regence Blue Shield and Premera Blue Cross); 4) Longitudinal financial and credit records from TransUnion; 5) Washington State bankruptcy records and 6) Medicaid enrollment records. Using this unique database that is updated annually, our first aim is to understand how the neighborhood characteristics, financial health, and clinical characteristics of commercially insured cancer patients have changed following ACA implementation. Due to expanded eligibility for commercial insurance under the ACA, we hypothesize that a greater proportion of cancer patients diagnosed post-ACA (2015-2022) live in disadvantaged neighborhoods, are financially fragile (lower credit, higher debt, delinquent payments), and have more comorbidities than pre-ACA (2009-2012) patients. Our second aim is to estimate post-ACA trends in out-of-pocket (OOP) cost exposure and underinsurance. We will characterize trends and variability in OOP exposure and risk of underinsurance for cancer patients across employer-based and ACA marketplace plans. Our third aim is to determine the extent to which commercially insured, cancer patients diagnosed after implementation of the ACA experience gaps in care and adverse cancer outcomes. In addition, we will measure the relationship between OOP cost exposure and adverse outcomes for these patients, defined as gaps in quality of care, financial hardship, and disenrollment from commercial insurance and/or enrollment in Medicaid following diagnosis. This unique study and ongoing collaboration with an external, community-based multi-stakeholder advisory group will provide crucial evidence to inform clinical and policy discussions aimed at identifying and reducing treatment gaps and adverse outcomes among commercially insured cancer patients in the post-ACA era.

NIH Research Projects · FY 2025 · 2024-07

Project Summary/Abstract The nematode C. elegans is a leading experimental and genetic model for aging, and both public and private institutions have made enormous investments in aging research in this system. Several lines of evidence show that the intestine is the major tissue affecting healthy lifespan in C. elegans. The intestine is likely to be a sensitive and early indicator of age-dependent cellular stress, as the intestine lacks an apoptosis pathway or stem cell population to remove or replace damaged cells. Intestinal nuclei have been shown to undergo progressive deterioration during aging, including the loss of entire nuclei. Old, abnormal intestinal nuclei show a loss of peripheral heterochromatin, a disruption of chromosomal organization, and the accumulation of intranuclear bodies that contain the protein lamin. Recent studies showed that some intestinal nuclei show abnormalities as early as the first two days of adulthood, and that these early abnormalities include the formation of lamin-containing nuclear bodies and the loss of peripheral heterochromatin. Because these defects occur early in development, they are likely to represent critical events that drive nuclear deterioration during aging, rather than indirect consequences of age-compromised cells. Some of the intranuclear lamin and loss of peripheral heterochromatin is associated with fat accumulation in the nucleus, called nuclear lipid droplets or nLDs. Recent studies have shown that diverse liver diseases in humans are associated with increased numbers of nLDs, and nLDs in C. elegans have been shown to increase during aging. nLDs in other systems show a complex relationship with fat, and nLDs in the C. elegans intestine appear to be correlated with changes in lipid storage that occur during reproductive development. This proposal investigates the origin of intranuclear lamin in intestinal cells, its relationship to nLDs, and the role of lipid storage. The research uses C. elegans tools for forward and reverse genetics to identify pathways that cause, or act to remove, the intranuclear lamin. The project analyzes specific roles for two genes which are involved in nLD formation and that are conserved in humans. The long-term goal of the project is to develop strategies that modulate these pathways to intervene in age-associated tissue deterioration.

NIH Research Projects · FY 2024 · 2024-07

ABSTRACT The efficacy of cell-based immunotherapies in treating solid tumors is hampered due to challenges pertaining to infiltration, function, and persistence of CD8+ effector T cells. The tumor microenvironment (TME) is designed to favor tumor survival and depletes key nutrients from the extracellular space, creating a hostile environment unamenable to CD8+ effector function. Regulatory T (Tregs) cells can thrive in the glucose-low TME due to their adaptable metabolic re- wiring which promotes survival of Tregs and facilitates their suppression of effector immune cells. High frequency of Tregs in the TME is associated with reduced response to immunotherapies, increased malignancy and poor clinical outcome. Metabolic dysfunction in the tumor not only supports the survival of immunosuppressive cells but drives an accumulation of metabolic byproducts that are secreted into the interstitial fluid of the TME where they can act as signaling molecules communicating with neighboring cells, driving suppressive phenotypes. Succinate is a critical mitochondrial metabolite that is secreted from the tumor due to an intracellular buildup in the hypoxic conditions of the TME. We have identified that extracellular succinate promotes the induction of Tregs from CD4+ T cells and further enhances the Th1-suppressing phenotype of Tregs. We propose that by inhibiting the effects of succinate on Treg induction and phenotype, targeting succinate signaling - specifically the succinate receptor (SUCNR1) - in CD4+ T cells and Tregs, we can increase the number of tumor-specific TCR-transduced CD8+ T cells that infiltrate the solid tumor thereby enhancing the anti-tumor response and reducing tumor burden. We will test this using tumors that have modified succinate levels (using CRISPR/Cas9), small molecule antagonists for the SUCNR1 on CD4+ T cells and WT and SUCNR1-/- CD4+ T cells, and comprehensively assess the effects of tumor-derived succinate on Treg induction in the TME. Utilizing our in-house high avidity TCRs that recognize the tumor antigens MAGE-A1 and PRAME, will subsequently develop a therapeutic strategy targeting succinate signaling to attenuate the suppressive effects of Tregs on tumor-specific CD8+ effector T cell anti-tumor responses, using SUCNR1-/- T cells and a small molecule inhibitor targeting succinate. We will examine this in vivo using advanced humanized mouse models (MISTRG) that support myeloid cells thus creating an immune diverse TME. We will simultaneously use a murine melanoma immune competent model (B16OVA OT-1) to interrogate the effects of succinate on immunosuppression on the TME. These studies will provide a novel and innovative therapeutic strategy for combating the resident and infiltrating suppressive Tregs in the TME and increase the efficacy of TCR immunotherapy outcomes in the solid tumor.

NIH Research Projects · FY 2026 · 2024-06

PROJECT SUMMARY We continue to lack vaccines for many pathogens of public health importance, including herpes simplex virus-2 (HSV-2), human immunodeficiency virus-1 (HIV-1), and tuberculosis (TB). In many infections of public health importance including HSV-2, the mucosal barrier is the portal of pathogen exposure, yet the regulation of mucosal immunity is incompletely understood. The broad objective of this proposal is to achieve a more in-depth knowledge of the regulation of mucosal immunity in the anti-pathogen immune response. Nature killer (NK) cells at mucosal sites are critical in controlling infections. NK cells are important for resolving HSV-2 infection by producing cytokines early after infection that activate adaptive immune responses, such as IFNg. Although NK cells are part of the innate immune system, there is evidence suggesting that NK cells can become long-lived memory-like cells and play an important role in secondary immune responses. NK cells remain functionally primed in the mucosal tissue up to 30 days after HSV-2 infection. During HSV-2 infection, NK cell activation is driven by inflammatory cytokines, and therefore must be subject to immune regulation to limit immunopathology. Regulatory T cells (Tregs) act by suppressing immunity and are crucial to maintaining peripheral tolerance and limiting tissue damage from excess inflammation. In the context of infection, there is evidence that Tregs unexpectedly also play a pivotal role in orchestrating an anti-pathogen response. Tregs are necessary during early HSV-2 infection to coordinate a productive anti-viral immune response in the vaginal tract (VT) in mice. However, the mechanism by which Tregs coordinate early antiviral innate immunity while limiting immunopathology remains poorly defined. While the link between NK cells and Tregs during infection is not well understood, there is evidence that the NK cell response is altered by the absence of Tregs. We previously published that Tregs are required for the proper homing of NK cells to the VT after HSV-2 infection. More recently, I have generated data that Treg depletion during HSV-2 infection increases NK cell maturation providing evidence that Tregs act in limiting NK cell response during viral infection. Thus, our rigorous prior research suggests a dual function for Tregs in modulating NK cell responses in which Tregs are required for both coordination of NK cell response and limiting excessive inflammation. However, the mechanisms and timing by which Tregs modulate NK cells within mucosal tissues during infection remain to be studied. This proposed work is essential for understanding the role of Tregs in coordinating and regulating NK cell function in mucosal tissues during viral infection. This understanding of mucosal immunology will be crucial to informing vaccine strategies to develop a balanced, robust, and tissue-specific immune response against viral infections, while still limiting immunopathology.

NIH Research Projects · FY 2025 · 2024-06

Abstract: Multiple myeloma (MM) is an incurable plasma cell tumor preceded by a defined preneoplastic state termed as monoclonal gammopathy of undetermined significance (MGUS). Strategies to effectively intercept/prevent clinical malignancy remain an unmet need and are the focus of this application. Changes in microbial communities have been extensively studied in the context of cancer outcomes and therapy. However, very few causal relationships between specific microbes and human malignancies have been defined, and suitable models to study these interactions are lacking. It is increasingly appreciated that MM/MGUS originate in the setting of prior chronic inflammation and B cell activation, but the underlying triggers remain unknown. This application involves three PIs (Dhodapkar, Flavell, Palm) with complementary expertise and a track record of productive collaboration. The application is based on exciting preliminary data describing the discovery of a commensal microbial species that directly engage several human MM-associated B-cell receptors. This is supported by data from new humanized models to study interactions between the human immune system and microbial communities in vivo. The proposal also utilizes biospecimens from MGUS patients treated with a short course of antibiotics to reduce pathogenic bacteria. Studies proposed in this application will characterize the full spectrum of human gut microbes that can potentially serve as antigenic triggers for human B cells and MGUS. It will also evaluate causal relationships between specific microbes and activation of human B cells as well as MGUS cells utilizing newly developed models. Finally, it will evaluate how microbial manipulation in MGUS patients can impact the inflammatory microenvironment known to be present and implicated in the pathogenesis of MGUS and its transition to clinical malignancy. Together, these studies may lead to novel approaches for immune-prevention of clinical MM by targeting the underlying precursor states such as MGUS. Insights from these studies may also have implications for interactions between specific microbes and human B cells, broadly impacting human autoimmunity and cancer immunotherapy.

NIH Research Projects · FY 2025 · 2024-06

Project Summary/Abstract Tumor progression – including resistance to therapy, metastasis, and recurrence – is responsible for the majority of cancer deaths. Understanding how cancer cells survive treatment, spread to distant sites, persist as dormant residual cells, and eventually recur is essential to improving the treatment of this disease. The long-term goal of my research group is to identify the pathways that regulate these processes in order to prevent or treat tumor recurrence. To achieve, this we are using conditional genetically engineered mouse (GEM) models of breast cancer that allow for the mechanistic dissection of the processes of dormancy and recurrence. Using these models, we have identified a functional role for the tumor suppressor Par-4 in regulating survival and recurrence of breast cancer cells after therapy. Par-4 is downregulated in recurrent tumors from three GEM models, and this downregulation is both necessary and sufficient for tumor recurrence. Similarly, in women with breast cancer, low Par-4 expression is associated with a poor response to neoadjuvant therapy and an increased risk of recurrence. We have characterized the upstream pathways that regulate Par-4 expression and function during dormancy and recurrence, as well as the downstream pathways that Par-4 regulates to inhibit dormant cell survival and recurrence. This proposal will build on this work to further explore the mechanism by which Par-4 acts as a tumor suppressor. The overarching hypothesis of the proposal is that Par-4 promotes cell death in part through inducing actomyosin contractility, and this contributes to its tumor suppressive functions. We will explore this hypothesis in the context of Par-4’s role in residual cell survival and the survival of invasive lobular cancer cells. Our work will reveal new information on how Par-4 functions to regulate dormant residual cell survival and may uncover novel opportunities for therapeutic intervention in eliminating residual cells and preventing recurrence.

NIH Research Projects · FY 2025 · 2024-06

ABSTRACT Relapse of leukemia occurs in nearly half of patients after hematopoietic cell transplantation (HCT). While there are a growing number of therapeutic options for leukemic relapse after HCT, we lack a clinical assay capable of simultaneously detecting disease and triaging patients to treatment which will most likely benefit them. In this application, we propose to rigorously evaluate and employ novel single-cell genomic methods and their accompanying computational approaches to detect disease at low levels, inform mechanisms of relapse, and direct future therapy. In Aim 1, we will optimize computational methods for detecting natural genetic variation between donor and recipient, a diagnostic maneuver we believe will improve the sensitivity of detecting malignant cells after HCT. We will then validate these methods using samples from patients in a retrospective fashion. To provide insight into mechanisms of relapse, we have developed a novel molecular approach for identifying perturbations in HLA and related genes, a common means by which leukemia evades the immune system after HCT. In Aim 2, we will validate this method in a manner that explores the lower limit of detection for HLA- perturbed cells. Finally, in Aim 3, we will build on decades of work using flow cytometry to assess the heterogeneity of myeloid neoplasms by integrating our single-cell molecular measures with cell-surface immunophenotype. Using this approach, we seek to define common molecular signatures in leukemia-initiating cells (LICs), a subset of cells in myeloid neoplasms thought to be a chemo-resistant reservoir of leukemia. The proposed studies will establish a new method for surveillance of residual disease after HCT, which we believe will provide a deeper level of confidence and insight into leukemic relapse post-transplant. We expect these studies to form the foundational preclinical data for a single-cell genomics clinical assay capable of risk-stratifying patients with early evidence of leukemic relapse after HCT.

NIH Research Projects · FY 2026 · 2024-05

PROJECT SUMMARY HIV persists despite decades of antiretroviral therapy (ART) because of a population of latently infected CD4+ T cells known as the HIV reservoir. The HIV reservoir is sustained by proliferation of infected CD4+ T cells, which do not express enough viral protein to be eliminated by HIV-specific immune responses. While proliferation of cells is a promising target for curing HIV and eliminating the need for lifelong ART, a comprehensive preclinical structure to develop a lymphocyte anti-proliferation therapeutic strategy does not exist. The optimal timing of anti-proliferative (AP) therapy is also unknown. Recent evidence suggests that CD4+ T cell proliferation plays a vital role in generating multiple proliferative clones of latently infected cells extremely early during untreated HIV infection. We developed a mathematical model which suggests that massive CD4+ T cell proliferation coincident with recovery from CD4+ lymphopenia, occurs during weeks 1-4 of primary HIV infection and is vital for generating much of the HIV reservoir. We hypothesize that effective AP therapy given during this critical three-week window will limit the volume and alter the clonal structure of the HIV reservoir. In Aim 1 of this application, Dr. Adam Spivak will test small molecular agents targeting CD4+ T cell proliferation given alone and in combination. A comprehensive library of immunomodulatory and chemotherapeutic agents with high therapeutic potential will be tested for their AP effects ex vivo on uninfected CD4+ T cell cultures, ex vivo on latently HIV-1 infected cells derived from human donors, and in vivo in uninfected rhesus macaques by measuring impact on CD4+ T cell turnover using deuterium water labeling. Finally, Dr. Joshua Schiffer will utilize mathematical models which capture drug pharmacokinetics and pharmacodynamics, as well as the underlying dynamics of CD4+ T cell subsets within the HIV reservoir, to optimize selection of single drug or combination anti-proliferative (AP) regimens for dosing of SIV infected animals in Aim 2. Drug regimens will first be ranked in a tabular form according to predicted potency. The most potent regimen with known safety in humans and lack of cell toxicity in Dr. Spivak’s ex vivo model will ultimately be selected for Aim 2. In Aim 2, Dr. Joseph Mudd will evaluate the effects of optimized AP agents on early reservoir formation dynamics in 24 SIV-infected rhesus macaques: 6 will receive ART alone between weeks 1-37 post infection; 6 will receive ART alone between weeks 4-40 post infection; 6 will receive ART between weeks 1-37 and optimized AP therapy between weeks 1-4 post infection; 6 will receive ART between weeks 4-40 and optimized AP therapy between weeks 1-4 post infection. Optimized AP regimens will be selected based on Dr. Spivak’s experimental data from Aim 1 coupled with Dr. Schiffer’s mathematical models. During ART, we will measure the in vivo AP therapeutic effect on 1) SIV reservoir volume with total and intact SIV DNA, 2) reservoir CD4+ T cell subset composition, 3) in vivo CD4+ T cell turnover with D2O labeling, and 4) SIV reservoir clonal structure using integration site sequencing, based on frequent longitudinal sampling of blood and gut tissues. After 36 weeks of ART, we will stop ART and monitor viral rebound for up to 4 months. We hypothesize that AP therapy between weeks 1 and 4 post infection will reduce total and intact SIV DNA and decrease reservoir clonality following 6 months of ART and increase time to SIV rebound after ART interruption.

NIH Research Projects · FY 2026 · 2024-05

PROJECT SUMMARY Native Hawaiian individuals represent one of the most underserved and understudied populations in US healthcare and medical research. However, this population is one of the fastest growing in the US and faces alarming long-standing health burdens, with approximately 30% higher risk of cardiovascular disease, 100% higher risk of diabetes and gout, and 400% higher risk of stroke compared to White individuals. Obesity and high body mass index (BMI) are known to increase risk for these conditions, and notably, an estimated 41% of Native Hawaiians are obese compared to 21% of White individuals. Native Hawaiians are characterized predominantly by Polynesian, European, and East Asian ancestries, and higher Polynesian ancestries in Native Hawaiians have been linked to higher risk of hypertension, type 2 diabetes, and heart failure, as well as higher BMI and subcutaneous and visceral fat, even after accounting for socioeconomic and smoking status. Despite Native Hawaiians sharing East Asian ancestry with Japanese individuals, the burden of some of these conditions are lower in Japanese Americans. As many of these health differences are cardiometabolic in nature, the objective of this research is to evaluate circulating metabolomic profiles in Native Hawaiians and Japanese Americans, given their complementary risk profiles, to illuminate the underlying mechanisms contributing to health burdens in these populations. This will be accomplished by establishing a large-scale high throughput metabolomic resource in 2,000 Native Hawaiian and 1,000 Japanese American participants from the Multiethnic Cohort. In Aim 1, we will conduct genome-wide association studies (GWAS) and evaluate genetic ancestry to investigate the contribution of genetics to metabolite variation. In Aim 2, we will conduct metabolome-wide association studies to identify metabolites associated with cardiometabolic and other health-related traits in Native Hawaiians. In Aim 3, we will investigate potentially causal metabolic mechanisms impacting health-related traits, leveraging the full sample of 5,660 Native Hawaiian and 24,485 Japanese MEC participants with GWAS data, along with GWAS summary statistics from >14,000 Pacific Islander and >214,000 Japanese participants across multiple studies. Findings from this investigation are expected to substantially improve our understanding of the underlying mechanisms contributing to cardiometabolic and other health-related conditions in Native Hawaiian and Japanese American populations, which will ultimately contribute to novel means to improve health. This study will also importantly bolster innovative epidemiologic research to address major knowledge gaps and health burdens in populations that are understudied and when included in research, often studied in aggregate, masking the ability to identify distinct etiologic factors.

NIH Research Projects · FY 2026 · 2024-04

PROJECT SUMMARY / ABSTRACT An effective HIV-1 vaccine will be one that elicits diverse anti-viral immune responses, including broadly neutralizing antibodies (bnAbs). Here, we focus on eliciting anti-CD4-binding site (CD4-BS) bnAbs, including VRC01-class bnAbs, through a guided immunization approach with specifically designed Env-derived immunogens. We previously reported on the design of a clade C Env-derived immunogen that activates naïve B cells expressing the bnAb precursors of VRC01-class antibodies in vivo. We also reported on the design of a second immunogen, derived from a clade B Env, that when administered as a 1st boost, increases the maturation of the emerging VRC01 B cells. Our proposal is based on our recent identification of two additional Env immunogens that complete the maturation of VRC01-class antibody responses towards their cross- neutralizing forms. Thus, after 4 Env immunizations of knock-in mice expressing elements of human VRC01 BCRs, we isolate VRC01-class antibodies that neutralize ~33% of heterologous tier 2 viruses. Here, our efforts focus on optimizing this immunization schema to improve the neutralizing breadth of the elicited VRC01-class antibodies. An important aspect of our proposal is that we will not limit our work to well-controlled knock-in mice, but transgenic mice that express diverse human VH/VL genes, as well. Because the B cell repertoire of these mice better reflects that of humans, we expect that our optimized immunization schema will not only lead to the generation of VRC01-class antibodies, as it does in the knock-in mice, but that additional classes of anti- CD4-BS antibody responses will also be elicited. A second important aspect of our proposal is that we will examine whether mRNA-based immunogen-delivery platforms accelerate the development of cross- neutralizing CD4-BS antibody responses.