La Jolla Institute For Immunology

NIH Research Projects · FY 2026 · 2022-04

PROJECT SUMMARY/ABSTRACT The Human Immunology Project Consortium (HIPC) was founded in 2010 to create a network of independent centers focused on measuring human immune responses with high-throughput systems immunology approaches coupled with detailed clinical phenotyping. We propose to develop a HIPC Coordinating Center (HCC) that will provide data more rapidly and effectively to the broader scientific community, and also serve the entire HIPC by increasing the value of the research performed at HIPC Centers. In parallel, the HCC will serve as a promoter of cross-HIPC collaborations through the organization of multi-center analysis projects and a centralized Portal to provide a space for the exchange of ideas. Specifically, the HCC will continue the development of shared data standards, provide a central Knowledgebase of study results, establish tools to visualize and analyze data, lead synergistic cross-center analysis efforts, provide a Portal to make the tools and data broadly accessible, and provide administrative support for the activities of HIPC subcommittees and management of the Infrastructure and Opportunity Fund (IOF) program. Our team has proven experience in running programs of similar scale and complexity. We also have the necessary familiarity with the HIPC network, with our team including current PIs of the Data Standards IOF project, the Signatures IOF project, clinical information capture, and the existing ImmuneSpace data portal. This proposal leverages this combined experience and infrastructure while, in parallel, implementing various significant and necessary improvements facilitated by the tighter integration and the 5-year time horizon of this dedicated HCC grant.

NIH Research Projects · FY 2025 · 2021-07

Project Summary Immune checkpoint blockade (ICB) therapy has demonstrated significant clinical benefit in late-stage patients with melanoma, renal cell carcinoma, head and neck cancer, Hodgkin lymphoma, bladder cancer, non-small cell lung cancer, gastric cancer, liver cancer, cervical cancer, Merkel cell carcinoma, and for all microsatellite- unstable tumors. A major limitation of ICB therapies targeting the CTLA4 or PD1 immune checkpoints is that a significant portion of patients will experience immune-related adverse events (irAEs), which can result in permanent or even fatal toxicity and discontinuation of life-saving immunotherapy. Compounding this problem is the fact that there currently exist no molecular modulators for ICB-driven irAEs. This R01 application examines circulating LPC 18:2 a novel small molecule modulator and therapeutic for irAEs by studying human cancer patients and relevant preclinical models of ICB-driven irAEs and tumor regression. The proposed aims will systematically i) examine association between LPC 18:2 and irAEs across multiple human cancer cohorts (e.g. melanoma, non small lung cancer, head and neck squamous cell carcinoma) and ICB therapies (e.g. anti- CTLA4 ipilimumab, anti-PD1 pembrolizumab and combination therapies); ii) examine relationship between plasma LPC 18:2 levels and ICB-driven tumor regression or natural autoimmune disease; iii) study the immunological mechanisms by which LPC 18:2 restrains ICB-driven irAEs and autoimmune colitis; iv) mechanistically probe novel effects of LPC 18:2 and LPC-G2A signaling on development and function of inflammatory neutrophils. This study uses a highly innovative approach leveraging cancer patient bio-sampling across multiple independent clinical trials with state-of-the-art rapid mass spectrometry profiling of small molecule metabolites and mechanistic studies. This is a collaborative study between a cancer immunologist and basic scientist at La Jolla Institute for Immunology and UCSD Moores Cancer Center, an analytical chemist at UCSD, a statistical epidemiologist at Cedars-Sinai Medical Center, clinical immune-oncology collaborators and experts on fundamental immunology and neutrophils. Validating LPC 18:2 as a therapeutic molecule for irAE toxicities addresses an urgent need at the clinical level to develop the very first therapies that can minimize risk, maximize benefit, and more accurately personalize ICB therapies for those patients who stand to benefit from cancer immunotherapy.

NIH Research Projects · FY 2025 · 2021-06

ABSTRACT The long-term goal of this project is to develop a dengue-Zika vaccine that provides protection against the four serotypes of dengue (DENV1-4) and Zika (ZIKV) viruses with maximal safety and efficacy. To date, flavivirus vaccine development has focused on the induction of neutralizing antibodies (nAbs), as they have been assumed to be the key mechanism for protection against natural infection. However, DENV and perhaps ZIKV are unusual in that weak Ab responses to vaccination or prior infection can induce antibody-dependent enhancement (ADE) of infection and pathogenesis during subsequent reinfections. In fact, DENV disease with severe sequalae has been documented in children given the only currently licensed DENV vaccine. Thus, the primary objective of this application is to develop an effective vaccine against DENV and ZIKV that cannot mediate ADE. We hypothesize that this vaccine will need to elicit both strong nAb responses and strong T cell effector responses that will counterbalance the presence of any ADE-mediating Abs, based on our work investigating the interplay between Ab and T cell responses to DENV and ZIKV. In particular, we have shown that CD8 T cells mediate cross- protection against heterotypic DENV and ZIKV infections, and that DENV vaccine-elicited CD8 T cells can prevent ADE. In addition, our preliminary data show that an RNA replicon-based vaccine expressing ZIKV nonstructural protein 3 elicits only T cell but not nAb responses and confers protection against ZIKV challenge in mice. Thus, we hypothesize that our combinatorial DENV-ZIKV vaccine expressing both Ab- and T cell-targeting proteins of DENV1-4 and ZIKV will produce humoral and cellular immune responses that provide robust, long-term protection against all five viruses. We will test this hypothesis by achieving the following Specific Aims: 1) To evaluate immunogenicity and efficacy of a DENV-ZIKV vaccine. 2) To determine the durability and mechanistic underpinnings of DENV-ZIKV vaccine-induced protective immunity.

NIH Research Projects · FY 2025 · 2021-05

ABSTRACT Recent years have witnessed a dramatic rise in interest towards cancer epitopes in general, and neoepitopes that encompass mutations arising in a given tumor in particular. Current lines of research examine how the epitope load in a given tumor relates to the success of checkpoint blockade treatments, and how to utilize epitope-based vaccines and adoptive transfer of epitope-specific T cells for personalized therapies. For these purposes, neoepitopes that are recurrently recognized in different individuals are of particular interest, which has also re-ignited interest in epitopes identified in classic tumor-associated antigens. Along with the interest in cancer epitopes, there is also interest in the TCRs and BCRs specifically recognizing them, as these have the potential to be used in therapeutic approaches, and they can aid in basic studies to infer the specificity of T cells or B cells characterized in single cell sequencing data. This resurgence of interest in epitopes has created a need to catalog and make accessible to the scientific community all epitope data, also linked to the biological, immunological, and clinical contexts. The ultimate goal is to come “full circle” and link epitope recognition and immunological readouts to clinical outcomes and treatment strategies alike. In parallel, there is an urgent need to develop resources for epitope prediction and analysis tools that provide access to predictive strategies and provide objective evaluations of their performance in the relevant biological, immunological, and clinical contexts. Recent years have also witnessed the publication of multiple original methodologies that reported sometimes impressive gains in the predictions of cancer epitopes. However, several of these studies were difficult to evaluate, because the methodologies and/or datasets were not fully available in a format that was readily executable. As a result, their performance could not be properly benchmarked on independent datasets. This is also because effective benchmarking on independent datasets requires the assembly of novel datasets of sufficient size and diversity. To overcome all of these information technology challenges, we propose to design and implement the Cancer Epitope Database and Analysis Resource (CEDAR), which will provide a freely accessible, comprehensive collection of cancer epitope and receptor data curated from the literature, and provide easily accessible epitope and TCR/BCR target prediction and analysis tools. As the cancer epitope data are curated, they will be used as a transparent benchmark of how well prediction tools perform, and also to develop new prediction tools for the analysis resource component of CEDAR. CEDAR will leverage our expertise from developing the Immune Epitope Database and Analysis Resource (IEDB), which is fully operational and widely used by researchers globally. CEDAR will directly complement other projects currently funded through the NIH ITCR program that provide resources and tools related to cancer omics data. Finally, we will engage in outreach activities to improve functions, user interfaces, and interoperability with other ITCR tools and promote the use of CEDAR in cancer research.

NIH Research Projects · FY 2025 · 2021-03

ABSTRACT Dengue virus (DENV) represents a major threat to global health. However, the precise role of the immune system in protecting against and pathogenesis of the four DENV serotypes, which share antigenic similarities and geographic ranges with each other and other closely related flaviviruses are poorly understood. In particular, antibodies can contribute to DENV pathogenesis by mediating antibody (Ab)-dependent enhancement of infection (ADE). This project focuses on defining the features of the anti-flavivirus Ab response that contributes to ADE vs protection using epidemiologically relevant mouse models in which DENV infection of mouse pups is enhanced by maternally acquired flavivirus Abs. Our published and new data demonstrate that flavivirus vaccination-infection combinations can promote either pathogenesis or protection. Our preliminary data also show that mice lacking T follicular helper (Tfh) cell responses are unable to induce DENV IgG response, and that mice treated with an agonistic Ab that stimulates OX40, a T cell costimulatory molecule belonging to the TNF receptor superfamily, exhibit a boosted DENV IgG response, suggesting that the magnitude of the Tfh response correlates with the level of Ab response to DENV. Therefore, we hypothesize that promoting Tfh responses will increase the production of broadly-neutralizing Ab (bnAb) responses that mediate protection and minimize ADE during DENV infection. We will test this hypothesis by using an RNA replicon-based vaccine platform that induces robust T cell and Ab responses in the following Specific Aims: (1) Determine how vaccination with different flavivirus antigens affects Tfh cell and Ab responses in maternal mice and susceptibility to DENV ADE in their offspring. (2) Test whether manipulation of immunization variables and candidate T cell costimulatory pathways boosts maternal Tfh cell and Ab responses and induces a protective response to DENV2 infection in offspring. These studies will provide critical insights into the factors and mechanisms that regulate ADE vs protective immunity to DENV2 infection. This knowledge is urgently needed to inform development of DENV vaccines that protect infants and young children, the highly vulnerable populations, and other flaviviral vaccines that are safe and effective worldwide, including in countries with co-circulation of 2 or more flaviviruses. The proposed work is based on our strong track record in investigating humoral and cellular immune mechanisms during flavivirus infections using state-of-the-art mouse models. The project will also benefit from our collaborators’ expertise in Tfh cells, T cell costimulatory molecules, flaviviral Ab response in humans, development of novel vaccine platforms, and genomics assays.

NIH Research Projects · FY 2024 · 2020-09

PROJECT SUMMARY This multi-PI proposal titled “SARS-CoV-2-reactive subjects” is written in response to ‘RFA-CA-20-039’ - tissue-resident memory T cells in healthy and cancer Research projects in SARS-CoV-2 Serological Sciences. Recent studies have shown that antibody responses to SARS-CoV-2 infection decline rapidly over time, implying a lack of durable protective humoral (B cell) immunity. Whether this is also true for cellular immunity (e.g., T cells) is poorly understood. It is well established that CD8+ TRM cells are the first line of defense in viral infections at mucosal/barrier sites. They are also known to protect hosts against homologous or heterologous re-infections. Our group was the first to show that TRM cells are pivotal players in driving effective anti-tumor immune responses in lung cancer, and that TRM cells are the primary cellular targets of anti-PD1 therapies. These key findings were possible because of the ongoing collaboration between Dr. Vijayanand, Dr. Ay, and Dr. Ottensmeier (Multi-PI). This team brings together experience in T cell immunology, single-cell genomics, bioinformatics, and cancer immunology. Our Multi-PI team has recently performed the first and largest single-cell RNA-seq and TCR-seq analysis of SARS-CoV-2-reactive CD8+ and CD4+ T cells (~300,000 single-cells) from COVID-19 patients. Here, to understand TRM responses to SARS-CoV-2, we will capitalize on a cohort of cancer (n=100) and non-cancer (n=100) patients, who will provide excess airway (nasal, oropharynx, larynx), lung and tumor tissue specimens obtained during routine surgery. In AIM 1, we will define the properties of SARS-CoV-2 reactive TRM cells from cancer and non-cancer patients with or without previous SARS-CoV-2 infection. We will perform combined single-cell RNA-seq and TCR-seq analysis of CD8+ TRM cells in the airways (nasal, oropharynx), lung, and tumor tissue. In parallel, by stimulating PBMCs with SARS-CoV-2 peptide pool, we will determine the transcriptomic and TCR sequence of SARS-CoV-2 reactive T cells. We will utilize this TCR sequence information to define the numbers and properties of SARS-CoV-2 reactive-TRM cells in mucosal and tumor tissues. Recent studies in non- exposed individuals (pre-COVID-19 pandemic) indicate pre-existing, circulating CD8+ T cells, with human coronavirus cross-reactivity. Here, we will measure the quantity and quality of pre-existing SARS-CoV-2 cross- reactive TRM responses in subjects without clinical or serological evidence of previous SARS-CoV-2 infection. In AIM 2, we will assess the impact of SARS-CoV-2 infection on anti-tumor and other anti-viral TRM responses. We will stimulate matched PBMCs (as above) with peptide pools targeting (i) common respiratory RNA viruses (influenza (FLU), RSV), (ii) persistent DNA viruses (CMV, EBV), and (iii) a tumor-driving virus (HPV) to define the TCR sequence of the respective virus-specific and tumor(HPV)-specific CD8+ T cells; we will utilize the TCR information to determine frequency and properties of other virus/tumor-reactive TRM cells in mucosal and tumor- tissue cells.

NIH Research Projects · FY 2024 · 2020-07

Abstract. The goal of cancer immunotherapy is to harness the immune system to destroy tumors in cancer patients. Two approaches have been successful in the clinic. (i) “Checkpoint blockade” therapies utilize blocking antibodies to inhibitory cell surface receptors or their ligands (CTLA4, PD-1/PD-L1) to deplete intratumoral regulatory T cells (Tregs) or to overcome a hyporesponsive state, termed “exhaustion” or “dysfunction”, that develops in CD8+ T cells that infiltrate solid tumors. However, only a subset of patients achieve complete remission, a problem that can potentially be countered by using combinations of antibodies to multiple inhibitory receptors. (ii) T cells expressing chimeric antigen receptors (CARs) that recognize tumor antigens are remarkably effective against hematopoietic cancers such as B-CLL (B cell chronic lymphocytic leukemia), but are not as effective against solid tumors, apparently because they become “exhausted” much like normal CD8 T cells responsive to standard peptide/MHC ligands. Here we propose a new strategy for increasing the effectiveness of CAR T cells attacking solid tumors. Some years ago, we discovered that TET (Ten-Eleven Translocation) enzymes are dioxygenases that use molecular oxygen, α-ketoglutarate (αKG) and reduced iron (Fe2+) to oxidize the methyl group of 5-methylcytosine (5mC) in DNA to 5-hydroxymethylcytosine (5hmC) and additional oxidized methylcytosines that are all intermediates in DNA demethylation. We have shown in mouse models that TET deficiency results in skewed cell lineage specification and enhanced signal-dependent cell proliferation in many cell types; impairs the function of T regulatory (Treg) cells by decreasing the stability of Foxp3 expression; and improves the ability of splenic CD4+ and CD8+ tumor-infiltrating T cells (TILs) to promote tumor regression. Moreover, Tet2-deficient mouse CD8+ T cells displayed cell-intrinsic expansion and skewing towards a central memory phenotype, both homeostatically and in response to viral infection; Tet2 deficiency in myeloid cells resulted in decreased immunosuppression by tumor-associated macrophages and myeloid-derived suppressive cells, resulting in more effective tumor regression by tumor-infiltrating T cells; and TET2-deficient CAR T cells promoted complete remission when administered to a patient with chronic lymphocytic leukemia. Here we will test the hypothesis that TET loss-of-function in tumor-infiltrating CD8+ T cells (CD8 TILs) improves tumor rejection. In Aim 1, we will examine the role of TET proteins in the expansion and function of CD8+ TILs. The metabolite L-2-hydroxyglutarate (L-2HG) is a potent inhibitor of TET enzymes and other αKG- and Fe2+- dependent dioxygenases. L-2HG levels are normally maintained at very low levels in cells by the enzyme L- 2HG dehydrogenase (L2HGDH). In Aim 2, we will assess the effects of L2HGDH depletion or L-2HG pretreat- ment on CAR TILs. In Aim 3, we will delineate the transcriptional networks involving TET enzymes in CD8+ TILs.

NIH Research Projects · FY 2026 · 2019-03

OVERALL COMPONENT SUMMARY Our LJI CCHI was first funded in April of 2019 and has been highly impactful in human immunology research in the past < 4 years, particularly in the areas of COVID and COVID vaccines. Crotty and Sette published the first major paper on virus-specific T cell and antibody responses in COVID cases ( > 3,000 citations) and went on to collaboratively publish several more of the most influential T cell, B cell, and immune memory papers on COVID and COVID vaccines. The adaptive immune system is important for control of most viral infections. The three fundamental components of the adaptive immune system are B cells (the source of antibodies), CD4+ T cells, and CD8+ T cells. The armamentarium of B cells, CD4+ T cells, and CD8+ T cells has differing roles in different viral infections, and in vaccines, and thus it has been critical to directly study adaptive immunity to SARS2 (SARS2) to understand COVID. The COVID pandemic has been a historic disaster, with over 1 million American deaths and millions of deaths and billions of infections around the world, counterweighted by the exceptionally efficient development of COVID vaccines which had remarkable efficacy and have saved over 15 million lives in less than two years. In 2023 COVID remains a major American public health problem and global health problem, with COVID being the #3 cause of death in the USA in 2022, and in the USA there were over 1.6 million confirmed new cases of COVID in the month of January 2023 alone. Improvements in controlling COVID remain somewhat impaired by our limited understanding of immune memory and upper airways immunity to SARS2. The overarching focus connecting the three Projects in this LJI CCHI renewal proposal is immune memory, with emphasis on COVID, highlighted by three overall LJI CCHI themes: (1) understanding immune memory in humans from blood samples, rich in complexities; (2) understanding human upper airways T and B cell biology and memory; and (3) COVID immunobiology, including breakthrough infections, differences between COVID vaccines, and immunity relatedness to other respiratory viral infections of humans. These three themes are explored in depth in Projects 1-3 and the Clinical Core.

NIH Research Projects · FY 2025 · 2018-08

Abstract The search for causal genetic variants associated with specific diseases among many variants identified by genome-wide association studies (GWAS) has been rejuvenated several times by the increase in throughput, resolution and the number/modality of experimental techniques that are broadly available. Advances in capturing cell-type-specific physical proximity among genomic regions also added a new dimension for this search by revealing the importance of 3D genome organization in interpreting the role of genetic variants in gene regulation. A number of combinations of these different techniques have proven useful in identifying a number of causal variants but many major challenges remain in our goal towards creating complete maps of genotype-phenotype associations for complex diseases. Our recent focus has been to address an important gap in the current knowledge of how genetic variants may impact 3D genome organization with or without a measurable impact on gene expression of the cell state/type that is available for molecular characterization. We have identified a number of genetic variants that associate with read coverage, strengths of specific loops and/or overall connectivity of large genomic regions. Leveraging our expertise in computational analysis and the newly established experimental component of our lab, we will address a number of questions emerging from our recent findings within the next five years. We will first define and characterize the role of genetic variants, which we found to be associated with specific chromatin loops and/or overall connectivity of regions harboring regulatory elements in specific human immune cell types. Next, we will perform long read-based assays and develop accompanying analysis methods to resolve allele-specificity and connection modality of multi-way interactions involving regulatory elements. Throughout the project period, we will continue developing computational methods for integrative, comparative and high-resolution analysis of conformation capture data. As we have done before, our methods development will be in alignment with biological questions we are trying to answer but with flexibility and generalizability in mind for their broad utility by other researchers.

NIH Research Projects · FY 2025 · 2017-06

Abstract DNA cytosine methylation (hereafter, DNA methylation) has a critical role in cell lineage specification as well as suppression of repetitive and transposable elements in the genome. DNA methyltransferases attach a methyl group to generate 5-methylcytosine (5mC); TET methylcytosine dioxygenases cause DNA demethylation by oxidizing the methyl group of 5mC to 5-hydroxymethylcytosine (5hmC) and beyond. We have shown that TET- deficient cell types display not only the expected increase in DNA methylation at promoters and enhancers, but also a paradoxical decrease in DNA methylation in heterochromatic regions of the genome. The consequences of these molecular features remain to be understood, but similar alterations in genome-wide DNA methylation patterns have been observed in cancer and aging. By studying the phenotypes of several mouse strains in which Cre recombinase was expressed either inducibly or developmentally in immune/ hematopoietic cell types, we showed that deletion of two or more Tet genes skewed cell lineage commitment in the relevant cell type, in a manner that correlated with changes in cell lineage commitment. More striking phenotypes, however, were that Tet2/3 fl/fl CD4Cre mice displayed massive TCR- dependent expansion of iNKT cells; and that Tet2/3 fl/fl Foxp3Cre mice developed a dominant proinflammatory phenotype observed in heterozygous female mice, in mixed bone marrow chimaeras, and in immunocompetent recipients injected with total CD4+ T cells from Tet2/3 fl/fl Foxp3Cre mice. This phenotype differs markedly from that observed in heterozygous Foxp3+/- females and in immunocompetent mice injected with Foxp3-deficient cells, which do not develop disease. In Aim 1, we will address the mechanisms underlying the striking expansion of Tet2/3-deficient iNKT cells by using adoptive transfer approaches in vivo and recently-developed cell culture systems that recapitulate the expansion in vitro. In Aim 2, we will ask whether the dominant autoimmune/ inflammatory phenotype of Tet2/3-deficient T regulatory cells requires, directly or indirectly, the decreased DNA methylation in heterochromatin observed in every TET-deficient cell type examined so far. Decreased DNA methylation in heterochromatin results in “heterochromatin dysfunction”, an aberrant cellular condition linked to autoimmune/ inflammatory disorders, cancer, aging, and neurodegenerative diseases, that stems from aberrant expression of transposable elements (TEs) and resulting DNA damage. DNA damage provokes “sterile inflammation”: activation of innate immune sensing pathways for RNA and DNA with consequent upregulation of type I interferons, interferon-induced genes and proinflammatory cytokines (e.g. IL-1b, IL-6, IFNg, IL-17) Our proposed experiments will add to our knowledge of how TET proteins influence T cell expansion and T regulatory function. More broadly, they will enhance our general understanding of the links connecting TET deficiency and TE expression with autoimmune/ inflammatory diseases, clonal hematopoiesis, a premalignant syndrome of older individuals associated with inflammation and cardiovascular disease, and cancer.

NIH Research Projects · FY 2025 · 2016-09

Abstract During the previous funding period, we made important strides in answering the fundamental mechanistic question of how TET2, DNMT3A and ASXL1 mutations give rise to clonal hematopoiesis (CH), cancer and inflammation. We performed biochemical and genomic analyses that linked TET deficiency to an unexpected loss of DNA methylation in heterochromatin. Given that DNMT3A and TET proteins have opposite biochemical functions, our finding of decreased DNA methylation in heterochromatin of both Dnmt3a KO and Tet2 KO cells is the only common feature that explains the unexpectedly greater disease severity of Dnmt3a KO, Tet2 KO (DKO) mice compared to mice with individual Dnmt3a or Tet2 gene disruptions. We also showed that CH- and cancer-associated mutations in ASXL1 result in reduced histone 3 lysine methylation (H3K9me2/me3) in hetero- chromatin, thus linking all three of the top proteins mutated in clonal hematopoiesis – TET2, DNMT3A and ASXL1 – to the fundamental process of impaired heterochromatin integrity due to reduced DNA or H3K9 methylation in heterochromatin. Interference with DNA or H3K9 methylation in heterochromatin has been known for decades to result in increased expression of transposable elements, genome instability and inflammation, all common features of cancer, inflammation and aging. Finally, we showed that OGT normally restrains TET activity in mESC, and that consequently, OGT deficiency increases TET activity genome-wide and results in genome-wide DNA demethylation. In this renewal application, we will ask how the TET-OGT interaction regulates DNA methylation, focusing on the protein-protein interactions and regulatory mechanisms that operate within TET- OGT complexes in mESC and hematopoietic-lineage cells. We will define how DNMTs and TETs cooperate to reduce DNA methylation in heterochromatin by examining DNMT3A redistribution and DNMT1/UHRF1 stability. We will explore the mechanism of how CH-associated mutations in ASXL1 lead to reduced H3K9 methylation in heterochromatin, again focusing on protein-protein interactions and regulatory mechanisms that operate within two distinct ASXL1-associated protein complexes that we have recently defined. We will identify individual members of TE families that are uniquely upregulated in TET2, DNMT3A and ASXL1-mutant cells, so as to determine how increased TE expression can have potentially indirect and stochastic effects on the expression of nearby genes by acting as promoters, enhancers or both. Lastly, we will perform CRISPR/Cas9 screens to elucidate how TET deficiency and ASXL1 mutations are linked to inflammation. Our studies will illuminate the mechanistically elusive connections between DNA and H3K9 methylation, clonal hematopoiesis, cancer, inflammation and aging, and improve our understanding of heterochromatin, a compartment of the genome that is very poorly understood.

NIH Research Projects · FY 2025 · 2016-08

This program will train postdoctoral research scientists in immunological mechanisms related to understanding how Autoimmune, Inflammatory, and Infectious Disease, and Cancer develop and potential ways to treat these diseases. The program at La Jolla Institute for Immunology (LJI) will involve 20 faculty members from LJI whose expertise is in immune cell function that relates to diseases such as diabetes, inflammatory bowel disease, multiple sclerosis, asthma, atopic dermatitis, Parkinson’s, influenza, dengue and zika virus, HIV, cytomegalovirus, tuberculosis, ebola, and multiple cancers. Each faculty mentor is internationally recognized in the field of immunology or immunological disease and each has substantial peer-reviewed research experience and track records. The program will be open to PhD, MD, and MD/PhD scientists interested in basic and applied research of the immune system. The training program will consist of a bench research laboratory experience of 12-24 months, under the close supervision of a mentor or several mentors working on problems relevant to understanding immunity and immunotherapy. The research backgrounds of the faculty mentors are diverse, but with common themes centered on the biology of the primary cells of the immune system, including T cells, B cells, NKT cells, macrophages, dendritic cells, neutrophils, and others. The training program will then represent an interdisciplinary approach where each trainee is exposed to cutting edge and state-of-the-art immunological techniques and systems for making fundamental and translational discoveries of the immune system. Our current and past trainees have excellent records of research accomplishments. New trainees will be recruited nationwide and efforts will be made to recruit and retain exceptional candidates. Trainees will be chosen on the basis of their prior academic performance, research experience, publications, interviews, and recommendations from supervisors. Trainees will participate in institutional seminars, journal clubs, courses, and immunology conferences designed to expand the breadth of their understanding of immunology and immunological disease. All trainees will be instructed in the principles of responsible conduct of research and scientific rigor and integrity. The goals of the program are to foster the development of well-rounded immunologists who can fill much needed academic positions, who can compete and be successful in obtaining independent research grants, and who can contribute to biotechnology and pharmaceutical approaches, aimed at treatment of immune-related diseases and improvement of public health.

NIH Research Projects · FY 2026 · 2014-08

ABSTRACT Modern immunotherapies have had a broad impact in clinical oncology. CAR T cell therapy has been dramatically successful against certain hematologic malignancies, and ‘immune checkpoint blockade’ targeting the PD-1/PD- L1 pathway or other T cell inhibitory pathways has proven to be an effective treatment for advanced solid tumors in a subset of patients. On the other hand, CAR T cell therapy has had only limited success against solid tumors, and many patients treated with immune checkpoint blockade either do not respond or experience tumor recurrence. One shared factor limiting the ability of these treatments to control cancer is that tumor-infiltrating T cells become ‘exhausted’. We need to understand immune cell exhaustion at a molecular level, both in mouse models and in humans, in order to design therapies that will be more effective at eradicating the original tumor and in fostering the development of tumor-specific memory cells that will prevent a recurrence. We discovered some years ago that the transcription factor NFAT— classically a main driver of T cell effector responses— also initiates T cell exhaustion. T cell receptor stimulation paired with effective costimulation activates NFAT and its transcriptional partner AP1, and drives the effector response. However, NFAT simultaneously activates a separate cell-intrinsic program that damps down T cell responses, with the relative strength of the two transcriptional programs depending on the context. In tumors, the hyporesponsiveness program is favored, leading to T cell exhaustion. Given that NFAT exhibits only modest affinity for its consensus sites in DNA when binding alone, there has always been the expectation that its role in exhaustion is supported by cooperative binding with unrecognized protein partners. In this project, we recently identified FOSL2, NR4A, TOX, STAT3, STAT4, and EOMES as physically adjacent to NFAT in CD8+ T cells expressing the hyporesponsiveness program and therefore as candidates to play the role of NFAT partners. Here, we plan to focus on one of these transcription factors, FOSL2, and address in detail the mechanisms by which it collaborates with NFAT to control the onset of exhaustion in mouse and human T cells. In Aim 1, we will delineate the contributions of FOSL2 to changes in gene expression during NFAT-driven CD8+ T cell hyporesponsiveness and during the early stages of CD8+ T cell engagement with a tumor in vivo. In Aim 2, we will investigate how FOSL2 acts in concert with NFAT at individual gene loci underlying exhaustion. In Aim 3, we will determine how FOSL2— once it has been upregulated by TCR signalling and NFAT, or by other physiological signals— can act on its own to promote the expression of certain exhaustion-related genes and to damp down the expression of IL2 and other effector cytokines. Our results will contribute to a deeper mechanistic understanding of key transcriptional mechanisms operating in mouse and human tumor-infiltrating T cells, and will provide a mechanistic context for engineering T cells that can circumvent T cell exhaustion in the clinic.

NIH Research Projects · FY 2025 · 2014-08

PROJECT SUMMARY While an enormous number of genetic variants have been associated with risk for human disease, how these variants affect gene expression in various cell types remains largely unknown. To address this gap as it relates to immune cells, as well as to identify which immune cell types are most susceptible to the effects of disease- risk variants, the DICE (Database of Immune Cell Expression, Expression quantitative trait loci (eQTLs) and Epigenomics) project was funded by the current R24 resource grant (R24AI108564). Although several autoimmune diseases like Systemic lupus erythematosus (SLE), display severe clinical manifestations in patients of Hispanic/Latino, Asian and African ancestry, the genetic risk factors that influence disease mechanisms have not been extensively studied, largely due to the exclusive focus of the initial genome-wide association studies (GWAS) on individuals of European ancestry. However, recent multi-ancestry GWAS meta- analyses that included individuals of diverse ancestries have identified many ancestry-specific disease-risk variants. To translate the biological significance of these GWAS-led discoveries in under-represented populations, high-resolution eQTL studies in immune cell types from individuals of diverse ancestries are needed to define the effects of disease-risk variants. While the current DICE cohort is more diverse (46% Europeans) compared to other large-scale eQTL studies, there remains a need to include equal representation from other globally prevalent ancestral populations. In Aim 1, we will generate transcriptomic and eQTL reference data in common and rare circulating immune cell types from equal numbers of male and female individuals of Hispanic/Latino, African, East Asian, South Asian and European ancestries (n=350; n=70/ancestral group) – DICE-Diversity resource project. We will perform single-cell RNA-seq in flow-sorted immune cell types and conduct single-cell-eQTL mapping to test associations between variants and gene expression. In Aim 2, we will define genes and cell types associated with autoimmune diseases in diverse populations, including minorities. To accomplish this objective, we will perform TWAS and colocalization analysis to integrate published multi-ancestry GWAS in autoimmune disease and eQTL datasets in immune cell types generated in this DICE-Diversity project. In Aim 3, we will make DICE data accessible and visible to the broader scientific community. We will expand our existing website to make current and newly generated experimental data and analysis tools available to the community. Overall, the renewal of the DICE project will greatly enrich this dataset with descriptions from individuals of different ancestries. The new DICE-Diversity database of transcriptomic and eQTL data for the human immune system should also facilitate mechanistic and functional investigations into the role of disease-risk variants emerging from multi-ancestry GWAS studies and eventually benefit health outcomes in diverse populations, including minorities.

NIH Research Projects · FY 2025 · 1991-08

ABSTRACT Modern immunotherapies have had a broad impact in clinical oncology. CAR T cell therapy has been dramatically successful against certain hematologic malignancies, and ‘immune checkpoint blockade’ targeting the PD-1/PD- L1 pathway or other T cell inhibitory pathways has proven to be an effective treatment for advanced solid tumors in a subset of patients. On the other hand, CAR T cell therapy has had only limited success against solid tumors, and many patients treated with immune checkpoint blockade either do not respond or experience tumor recurrence. One shared factor limiting the ability of these treatments to control cancer is that tumor-infiltrating T cells become ‘exhausted’. We need to understand immune cell exhaustion at a molecular level, both in mouse models and in humans, in order to design therapies that will be more effective at eradicating the original tumor and in fostering the development of tumor-specific memory cells that will prevent a recurrence. We discovered some years ago that the transcription factor NFAT— classically a main driver of T cell effector responses— also initiates T cell exhaustion. T cell receptor stimulation paired with effective costimulation activates NFAT and its transcriptional partner AP1, and drives the effector response. However, NFAT simultaneously activates a separate cell-intrinsic program that damps down T cell responses, with the relative strength of the two transcriptional programs depending on the context. In tumors, the hyporesponsiveness program is favored, leading to T cell exhaustion. We recently identified TOX- and NR4A-family transcription factors as genes induced by NFAT that cooperate with NFAT to further the exhaustion program. It has been thought that exhaustion is a gradual process, but in fact we have demonstrated that TOX mRNA and TOX protein— along with other markers of exhaustion— are sharply induced in tumor antigen-specific CD8+ T cells almost immediately when they encounter a tumor. This means that CAR T cells or expanded tumor antigen- specific T cells are immediately at risk when they are infused into a patient, perhaps explaining the requirement for transferring large numbers of cells, and accounting in part for the limited success of the therapies. Here, we plan to address the fundamental transcriptional mechanisms controlling the onset of exhaustion, as a background for eventually circumventing this problem in the clinic. In Aim 1, we will define the ensemble of transcription factors and, by extension, the cellular signalling pathways that drive TOX expression and lead to exhaustion. In Aim 2, we will probe how TOX then furthers the exhaustion program by directly controlling key exhaustion-specific genes. In Aim 3, we will address the very practical question whether TOX protein levels in tumor-infiltrating lymphocytes can be titrated to prevent or reverse exhaustion, taking an approach that could in principle be developed further for clinical use. Our results will contribute to a broad mechanistic understanding of key transcriptional mechanisms operating in mouse and human tumor-infiltrating T cells.