While many strides have been made in the study of human immune responses, numerous knowledge gaps still remain. Many infections and immune-mediated diseases are localized to specific tissues, or organs but little is known about tissue/organ-specific immunity. A major overall goal of this CCHI program to this end is to understand memory CD8 T cells differentiation in the blood, tissues and organs. We have added Dr. Donna Farber to our CHHI-EVC team this cycle. She has established a unique human tissue resource enabling acquisition of blood and various tissues from deceased organ donors, we will therefore analyze virus-specific memory CD8 T cells in various tissues of deceased organ donors and address questions regarding the anatomic distribution and the epigenetic, transcriptional and phenotypic profile of virus-specific CD8 T cells elicited by live- attenuated virus vaccines and with minimal risk of antigenic re-exposure. The second major goal of this program is to elucidate the molecular mechanisms of “trained immunity” after YFV vaccination and viral infection to and harness this knowledge for future development of new classes of vaccines and immunotherapies. We will address the following questions. To what extent is so called “innate memory” caused by the effects of an ongoing adaptive immune response (for example, via paracrine signaling), versus a cell intrinsic property of innate cells, similar to the classic phenomenon of immune memory exhibited by memory T or B cells? Is there an enhanced response of DCs and monocytes, (similar to a memory response in the adaptive immune system), during secondary vaccination or infection? If so, what are the cellular and molecular mechanisms involved? Finally, the third overall goal is to identify signaling and transcription factor (TF) networks associated with T memory cell differentiation and survival and quantify how these networks change with age. This will be achieved in the following projects: Project 1: Immune memory (Ahmed, Hellerstein, Farber); Project 2: Innate immunity (Pulendran, Hellerstein), and Project 3: Immune senescence (Goronzy, Greenleaf). Supported by the following Core A: Administration (Ahmed); Core B: Single cell and integrative genomics (Bosinger, Greenleaf); Core C: Clinical and biostatistical (Edupuganti, Kulkanya, Yu).
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Our U19 Center consists of three Cores and three Projects:
CORE A: ADMINISTRATIVE CORE
AHMED, RAFI, Core Lead
The major objectives of the Administrative Core of the Cooperative Center on Human Immunology at the Emory Vaccine Center (CCHI-EVC) are to institute streamlined processes facilitating productive interactions among the investigators of the renewal grant application entitled “Vaccine Induced Immunity in the Young and Aged”. In order to achieve these objectives and to ensure a seamless operation of the Core, we will establish an Operations Office, which will be responsible for carrying out the following activities/functions at the CCHI-EVC: 1) Provide the infrastructure for the overall management and co-ordination. 2) Facilitate and promote communication and interaction among the PIs/investigators (Research Projects, Technology Development Project and Cores) by conducting regular teleconferences/face-to-face meetings, annual meetings, as well as seminars/symposia on human immunology of bio-defense pathogens. 3) Resolve all or any potential conflicts that might arise within and outside of CCHI-EVC by implementing recommendations of the Conflicts Resolution Group. 4) Provide fiscal/scientific oversight, review and consolidate yearly progress reports sent to the NIH. 5) Protect intellectual property rights of CCHI-EVC investigators and to execute material transfer agreements. 6) Facilitate data management/sharing among investigators in and out of CCHI-EVC. Thus, the Administrative Core is organized to foster a high degree of synergy and productive interaction among investigators for achieving success in the overall objectives of CCHI-EVC.
CORE B: SINGLE CELL AND INTEGRATIVE GENOMICS CORE
BOSINGER, STEVEN EDWARD, Core Lead
The Single Cell and Integrative Genomics Core will provide support for individual Projects by performing assays requiring specialized technology and offering unique bioinformatics methodology. More specifically, Core B will be responsible for the following work: (i) conducting bulk and single-cell RNA-Seq profiling; (ii) running bulk and single-cell ATAC-Seq assays to assess chromatin accessibility, and (iii) providing computational expertise and bioinformatics for the analysis of Core B generated data that is beyond the capabilities of individual Projects. Core B is led by Dr. Steven Bosinger at Emory University, with Dr. Will Greenleaf at Stanford as a key Co-Investigator. Core B will be physically located at both Emory and Stanford. The Emory site of Core B (Bosinger) will conduct bulk and single-cell RNA-Seq library preparation and sequencing for the experiments described in each project. The Stanford site of Core B (Greenleaf) will be responsible for ATAC-Seq library preparation on bulk and single-cell samples. Both sites will take advantage of novel liquid handling platform work-flows to enable single-cell RNA-Seq and sc-ATAC-Seq library generation to be performed in a high-throughput, cost-effective manner. Hence, Core B will be able to obtain information on the epigenetic landscape of individual immune cells longitudinally after vaccination at a resolution and scale that has previously not been feasible. Core B will also provide unique expertise in analyzing sc-ATAC-Seq data that will take advantage of the availability of the sc-RNA-Seq data from matching samples. By integrating single-cell RNA-Seq and ATAC-Seq data, these analytical approaches allow for epigenetic states that predict transcriptional states to be identified with accuracy. Additionally, Core B, has developed methodology to construct lymphocyte differentiation “trajectories” to order epigenetic and transcriptional changes at different stages of development in single cells. This analytical approach will be applied to assess lymphocyte differentiation following vaccination and in aging. In summary, the technological expertise offered by Core B will allow for high-quality single-cell genomic data to be generated in a highly efficient and cost-effective manner. More importantly, the integrated analytical pipelines developed and offered by Core B will be central to attaining the research aims of each Project, specifically in building high resolution models of genetic states that are imparted on lymphocytes during the acquisition of memory or senescence and identifying factors that dictate these long-term fates.
CORE C: CLINICAL AND BIOSTATISTIC CORE
EDUPUGANTI, SRILATHA, Core Lead
Dengue and yellow fever viruses (YFV) cause some of the most important mosquito-borne diseases with extensive morbidity and mortality around the world. Yellow fever virus causes an acute hemorrhagic fever complicated by hepatitis, renal failure, coagulation abnormalities, and in severe cases, death. Currently, there are large outbreaks of yellow fever in countries in West Africa and South America. A live, attenuated yellow fever vaccine (YFV-17D) has been in use since the 1930s and is highly efficacious in preventing yellow fever. It provides long-term immunity for over 30 years and up to a life time. The FDA licensed yellow fever vaccine allows us an opportunity to study the underlying immunological mechanisms that confer long term protective immunity in humans. Dengue virus infection is the most prevalent mosquito-borne infectious disease in the world. There are four distinct, but closely related serotypes of dengue virus (DEN-1, DEN-2, DEN-3 and DEN-4). Recovery from infection by one serotype does not provide protective immunity to another. The incidence of dengue fever and a more severe form of dengue known as dengue hemorrhagic fever have increased dramatically worldwide with 40% of world’s population in over 100 countries being at risk. Understanding protective immune responses and long-term immunity in natural infection is key for vaccine development. Knowledge gained from these studies will inform the immune responses needed for a safe and effective dengue vaccine. In the past and current CCHI funding cycles, the Clinical Core has been a vital component of the Emory CCHI program and continues to support the CCHI scientific agenda for over 10 years. The Core has three units: (1) Hope Clinic Unit at Emory University, Atlanta, GA (2) Dengue Clinical Unit at Siriraj Hospital of Mahidol University, in Bangkok, Thailand; (3) Statistical Unit at Emory University, Atlanta, GA. In this proposal, we plan to continue to perform novel innate and cellular immunity studies along with state-of-the art single cell and integrated genomics to advance our fundamental understanding of immune memory, innate immunity, immune senescence. The Hope Clinic Unit will continue studies with the YFV vaccine clinical studies to provide appropriate specimens for Projects 1, 2 and 3. The Dengue Clinical Unit at Siriraj Hospital in Bangkok, Thailand will continue with the dengue studies from acutely infected children and adults. The innate immunity and immune senescence work will continue at Stanford University.
PROJECT 1: IMMUNE MEMORY
AHMED, RAFI, Project Lead
The cardinal properties of memory CD8 T cells are their longevity, rapid elaboration of effector functions and the ability to proliferate upon re-exposure to the pathogen. We have been addressing fundamental questions about the origin and differentiation of human memory CD8 T cells using the live attenuated yellow fever virus vaccine (YFV-17D) and have made substantial progress during the current cycle of funding. Using in vivo deuterium labeling to mark virus specific CD8 T cells, we have shown that the memory pool originates from CD8 T cells that divided extensively during the first two weeks after infection and is then maintained by quiescent cells that divide less than once a year. Although these long-lived YFV specific memory CD8 T cells did not express effector molecules and had a transcriptional profile similar to naïve CD8 T cells, their epigenetic landscape resembled that of virus specific effector CD8 T cells. This open chromatin profile at effector genes was maintained in memory CD8 T cells isolated even a decade after vaccination, indicating that these cells retain an epigenetic fingerprint of their effector history and remain poised to respond rapidly upon re-exposure to the pathogen. These findings have prompted us to ask several new questions about human memory CD8 T cell differentiation. Specifically, what are the transcriptional and epigenetic changes taking place at the single cell level during this effector to memory cell transition? Can we identify fate-permissive and fate-locked YFV specific effector CD8 T cells and follow their differentiation trajectories longitudinally? Is the memory differentiation paradigm that we have defined with our YFV-17D studies generalizable to other acute viral infections? Will memory CD8 T cells after different acute viral infections look the same or will they be different? Does childhood vaccination result in the same memory differentiation program as vaccination of adults? Finally, how are tissue resident memory cells distributed, and how do the phenotype, transcriptional, and epigenetic signatures of these cells compare to the long-lived memory CD8 T cells we have found in the blood. To address these questions the following Specific Aims are proposed: Aim 1. Mapping the differentiation trajectories of virus specific effector CD8 T cells as they transition to long-lived memory. Aim 2. To characterize memory CD8 T cells responses to childhood vaccines and acute viral infections. Aim 3. To analyze virus-specific tissue-resident memory CD8 T cells generated by vaccination and infection.
PROJECT 2: INNATE IMMUNITY
PULENDRAN, BALI, Project Lead
Immunological memory is a hallmark of antigen-specific T and B lymphocytes. In contrast, the innate immune system is known to launch rapid, non-specific effector responses, which are short-lived. However, recent studies have proposed a form of immunological memory in the innate immune system, where innate cells can “remember” a pathogen encounter for several weeks to months. This phenomenon of “trained immunity” has been documented for NK cells, but less is known about its role in monocytes and dendritic cells (DCs). Innate memory has been suggested to be mediated via epigenetic changes in myeloid cells, but there are several fundamental questions about the mechanisms of innate memory. In this proposal, we will address the following questions in the context of vaccination with the live attenuated yellow fever vaccine 17D (YFV-17D) or acute dengue viral infection in humans, and in mechanistic studies in mice: 1. Unlike memory T and B cells, most DC and monocyte subsets are believed to have a relatively short lifespan of a few days. So how can epigenetic changes acquired by such short-lived cells mediate innate memory? Can subsets of myeloid-derived cells persist for several weeks after infection or vaccination? 2. To what extent is so called “innate memory” caused by the effects of an ongoing adaptive immune response (for example, via paracrine signaling), versus a cell intrinsic property of innate cells, similar to the classic phenomenon of immune memory exhibited by memory T or B cells? 3. Is there an enhanced response of DCs and monocytes, (similar to a memory response in the adaptive immune system), during secondary vaccination or infection? If so, what are the cellular and molecular mechanisms involved? We will address these questions in the following aims: Aim 1. Determining the innate response and regulatory landscape of DCs and monocytes in response to YFV-17D vaccination and dengue infection in humans. Aim 2: To determine the turnover rates of DCs and monocytes in response to YFV-17D vaccination in humans, using heavy water labeling. Aim 3: To define the mechanisms of innate memory induced by YFV-17D and adjuvants. Successful completion of these aims will further our mechanistic understanding of the phenomenon of innate memory, and offer novel strategies inducing broad and durable protection against diverse pathogens.
PROJECT 3: IMMUNE SENESCENCE
GORONZY, JORG J, Project Lead
The ability of the adaptive immune system to develop immunological memory is central to immune health and is the foundation for the great success of vaccination programs in preventive medicine. With increasing age, the proficiency for forming immunological memory dramatically declines, resulting in incomplete protection after vaccination. The cause for this decline is multifactorial, with an inability in maintaining a naïve T cell compartment as well as a failure of T cells to respond and differentiate upon antigenic stimulation. In our current studies, we have found that epigenetic signatures are sensitive and robust to identify T cell-intrinsic changes as they occur with aging and allow conclusion on the transcription factor networks involved. Age-associated changes, both at the transcriptional as well as epigenetic level, fall into two dimensions; failure to maintain stem-like features resulting in the initiation of differentiation programs, and defects in cell maintenance pathways. Interestingly, CD8 T cells are more sensitive to these changes than CD4 T cells. In Aim 1, we will use this difference between CD4 and CD8 T cells to examine the influence of age on T cell homeostasis. We will define naïve T cell population heterogeneity at the single cell level and examine the hypothesis that transcription factor networks in CD4 T cells are protective to aging. In Aim 2, we will study the influence of age on TCR signaling and analyze how TCR signal strength and age-associated changes in microRNA expression shape activation-induced modifications in the epigenome of T cells from young and older individuals. Aim 3 will examine the epigenetic basis for the preservation of functional long-lived memory cells. Based on longitudinal studies of chromatin accessibility in antigen-specific CD8 T cells, we have inferred transcription factor networks that characterize memory cells years after yellow fever vaccination. We will now extend these studies to address the hypothesis that transcription factor networks in memory T cells change with age. We will analyze CD4 and CD8 memory T cells to different viruses, which are known to behave differently to aging, such as varicella zoster and cytomegalovirus. In addition, we will examine in single cell studies whether aging influences the cellular heterogeneity in these virus-specific memory T cells. We will compare our results in the aged host to those generated in Project 1 on tissue-resident T cells or CD28+ and CD28- effector and memory T cells after yellow fever vaccination. Collectively, our studies will define the epigenetic landscape in antigen-specific T cells that confer effector or long-lived memory function and relate epigenetic changes to mechanisms relevant to T cell aging.
