Tissue and organ-specific human B cell immunity Project Summary Experiments conducted using genetically modified mice and model pathogens or antigens have shaped our current understanding of adaptive immunity. From these studies, it is clear that both B and T cell immunity is exquisitely tailored to the pathogen or antigen. Furthermore, the data show that adaptive immune responses in lymphoid and peripheral tissues, which are found in both mucosal and systemic sites, can differ dramatically. Thus, we now know that studying immunity in a single lymphoid tissue to model antigens, while informative, does not reveal the full complexity of the adaptive immune response. This lesson, learned using mouse models, can also be applied to human immune responses. To date, the vast majority of human immune responses have only been queried in the blood. This approach has not only limited our analysis to the lymphocytes that are in circulation but has also restricted us to examining a small fraction of the cells (typically 1-2%) that are in circulation at a given time. This “limited sampling approach” has also impaired our capacity to examine antigen- specific lymphocytes as these cells are rare populations, even during an ongoing immune response, and are largely undetectable under homeostatic conditions. Thus, we know exceedingly little about the phenotype, molecular programming, lifespan and function of human immune cells that reside primarily in tissues under homeostatic conditions. Moreover, we know essentially nothing about the clonal connections between antigen- specific cells in the different tissues of the human body. These represent fundamental gaps in our basic knowledge of the human immune system and reduce our ability to design new vaccines to prevent emerging infections, to create immunotherapies that can be used to treat cancer, autoimmunity and chronic disease and to develop tolerance-inducing regimens that will improve transplantation outcomes. Therefore, the overall goal of this U19 Program is to determine the molecular and functional relationships between human memory B lymphocyte and antibody secreting cell (ASC) populations that are found in pulmonary, intestinal and adipose tissues under homeostatic conditions. This U19 Program, which is composed of three Projects and three scientific Cores, will test our central hypothesis that antigen-specific, antigen-experienced human tissue-residing B cells are heterogeneous with respect to phenotype, transcriptome, breadth of reactivity, and function. We expect our studies to reveal the identity of novel subsets of antigen-experienced B cells exhibiting unique tissue- specific “signatures”. This outcome will inform future studies of fundamental basic human immunobiology and is likely to influence future translational and clinical studies, as B cells specific for the viral antigens and bacterial cell wall components are important targets for vaccination and are required for host defense against a number of different Biodefense Category A and C pathogens.
Our U19 Center consists of five Cores and three Projects:
LUND, FRANCES E., Core Lead
The immune system plays a critical role in protecting us from infection and resolving or repairing tissue injury following infection or other environmental insults that cause damage. The immune system is also a major contributor to chronic inflammatory diseases that drive health care costs and morbidity and mortality in the U.S., including cardiovascular disease, diabetes, autoimmunity, asthma, transplant rejection and many others. Over the last three decades we have learned much about how immune cells develop and mature, how immune cells are activated and differentiate into “effector” cells, how immune cell memory is initiated and maintained and how immune cells contribute in a functional way to immune-mediated protection and damage. In this U19 Program, we are particularly interested in B lineage cells that contribute to immune function by presenting antigen and activating CD4 T cells, by altering the immune microenvironment through the production of cytokines and chemokines and, most importantly, by differentiating into short- and long-lived antibody producing plasma cells (ASCs) and long-lived memory B cells. These cells are responsible for the humoral arm of the adaptive immune system and are key to host defense against most viral and bacterial pathogens. However, antibodies produced by ASCs are also responsible for much of the tissue damage associated with autoimmunity, asthma and transplantation. Many of our insights into the protective and pathologic functions of B lymphocytes and ASCs have come from genetically modified mouse model studies and, although these studies have been very informative, it is clear that mice and humans are not identical and that there is a need to better understand how human B cells contribute to immune (dys)function. Our studies of human B cells are still quite rudimentary – largely because the studies have been limited to the easily accessible circulating blood compartment. This is the equivalent of studying the tail of the elephant without seeing the whole elephant – we miss most of the complexity and heterogeneity of B cells when we only examine a tiny fraction of the cells that are present in the body. In this U19, we plan to move beyond the elephant tail as our goal is to comprehensively examine the molecular, cellular and functional properties of human B cells that reside specifically in the less accessible human tissues and organs. The major objective of Core A is to provide oversight and support for the overall Program and the individual projects and cores. We will meet this objective by: (i) providing all projects and cores with scientific, administrative, regulatory and financial oversight; and (ii) organizing scientific interactions, including the monthly research in progress meetings for the Program scientists, the annual retreat with the scientific advisory board, the annual meeting with other Cooperative Centers on Human Immunology groups and the Human Immunology themed session at the Southeastern Immunology Symposium. Through these activities, Core A will ensure the overall success of the Program.
HUMAN TISSUE COLLECTION, PROCESSING AND REPOSITORY CORE
NELLORE, ANOMA, Core Lead
Bridges, Jr., S. Louis, Co-Core Lead
Circulating lymphocytes are easy to access and therefore have been intensively studied in human beings. In contrast, much less is known regarding the biology of tissue-resident human lymphocytes. This problem exists because of limited access to sufficient quantities of human tissues. The overarching goal of this U19 Project is to advance understanding of humoral immunity by evaluating B cells in eight different human tissues. Three projects and three scientific cores will collaborate to examine the responses of B cells in these tissues against bacterial, viral, and transplant antigens. Core B’s objective is to eliminate the challenges associated with human tissue access in support of the U19 goals. The organizational and operational structure of Core B is innovative because a team with broad expertise (surgical, immunologic, cell biology, and biorepositories) will be aligned with the Alabama Organ Center (AOC). The AOC manages deceased organ donors for the purposes of tissue transplantation and is on the same campus as Core B. This structure will enable the collection of tissues from approximately 19 human donors per year. Further, Core B will isolate, quality control and store leukocytes in a biorepository. The rationale for this approach is that it will not only increase human tissue access, it will standardize the isolation and storage of high-quality leukocytes. The repository component will enable the projects and cores of this U19 to search this collection to find the ideal samples for each experiment. This setup also facilitates the collaborative aspects of this U19 by enabling all projects to understand which samples exist so that sharing can be prioritized to maximize sample value. In Specific Aim 1, we will collect tissues from deceased organ donors via the AOC. The objective of Specific Aim 2 is to isolate and cryopreserve leukocytes from the tissues. Lastly, Specific Aim 3 will establish a searchable biorepository of these samples with which to support the research objectives of the U19. The activities of Core B are significant because they will eliminate barriers that have prevented access to tissue-resident lymphocytes, and they will enable the comparison of antigen-specific B cells in multiple tissues collected from the same individual, as well as comparison to other individuals. Many of these investigations will be entirely novel.
B CELL RECEPTOR REPERTOIRE, CLONING AND EXPRESSION CORE
RANDALL, TROY D, Core Lead
King, Rodney G., Co-Core Lead
B cells express a widely diverse repertoire of B cell antigen receptors (BCRs) that can be further diversified by isotype-switching of heavy chain constant regions and by somatic hypermutation (SHM) in the antigen-binding domains of both heavy and light chains. B cells responding to protein antigens, such as viral antigens or transplant antigens, are often expanded in the germinal center, which stringently selects for high affinity memory B cells and antibody-secreting cells (ASCs). In contrast, B cells responding to carbohydrate antigens, such bacterial capsule components or blood group antigens, rarely enter the germinal center, but are nonetheless highly-selected based on reactivity and cross-reactivity to various antigens. The processes of clonal expansion and selection leads to the formation of B cell lineages that are related by BCR sequence and antigen-specificity. Importantly, the characteristics of the BCR (V gene use, SHM, isotype, affinity, cross-reactivity) often determine the functions of individual B cells, the tissues in which they reside, and the effector activities of the antibodies they produce. However, we have a limited understanding of antigen-specific B cells in the non-lymphoid tissues of humans. Therefore, the projects of this U19 will characterize B cells specific for different types of antigens (glycans in Project 1, viral antigens in Project 2 and xenoantigens in Project 3), obtained from lymphoid, mucosal and adipose tissues of normal human donors. Importantly, each project will determine the biochemical and molecular characteristics of antibodies made by individual B cells, determine the clonal relationships between antigen-specific B cells in different locations and use cytometric bead arrays to characterize the specificities, cross-reactivities and affinities of antibodies made by individual B cells. In order to achieve these goals and accelerate the research activities of each project, Core C will sort individual B cells from selected populations into 384-well plates, amplify VH and VL gene segments, clone the amplicons into IgG expression vectors, transfect the vectors into eukaryotic cells and produce recombinant monoclonal antibodies. Core C will also perform BCR heavy chain repertoire sequencing on sorted memory B cells and ASCs from the same tissues/donors used by the projects to sort antigen-specific B cells. Finally, Core C will develop cytometric bead arrays to enable each project to assay the reactivity profiles of serum samples, culture supernatants and monoclonal antibodies. Together, these activities will promote synergy and cooperation between projects and will accelerate the pace of research by centralizing procedures that require specialized expertise and instrumentation and by standardizing reagent development and production for each of the individual projects.
DATA ANALYTICS AND BIOINFORMATICS CORE
ROSENBERG, ALEXANDER, Core Lead
Hypothesis-testing studies designed to understand the phenotypic, molecular and functional differences between antigen-specific B cell subsets found in human lymphoid and peripheral mucosal tissues that are described in this proposal will rely on several ‘omics and other data-rich approaches and corresponding analyses to achieve their goals. To support use of these platforms and their associated data analyses, we propose to provide unified and integrated data and informatics services in Core D, the Data Analytics and Bioinformatics Core. The core will cover three broad areas as reflected by the Specific Aims: data management, data processing pipelines, and collaborative, downstream analysis. In Specific Aim 1, we propose to oversee and implement systems and processes to manage donor demographic and sample processing metadata, resulting data sets (raw through processed) and analysis provenance with all the appropriate linkages. This will benefit the Program by creating infrastructure that can be efficiently used by all the Projects and Cores as well as promoting good data stewardship practices which in turn, supports reproducibility. In Specific Aim 2, we propose to implement standardized workflows to cover all of the high-throughput platforms used in this Program (by one or more Projects): RNA-seq, single-cell RNA-seq, B cell receptor sequencing (for Sanger sequencing as well as for repertoires, by next-generation sequencing), and multi-parameter flow cytometry using semi-automated approaches. This will benefit the Program by standardizing primary data processing across the Projects and will promote comparability of results. In Specific Aim 3, we will provide collaborative downstream bioinformatics analytical and statistical support for all three Projects. This will benefit the Program by serving as a resource that all Investigators in the Projects can access for using data analyses to address their hypotheses, and by centralizing this function, we will economize this support as the analytical needs and methodologies of the Projects will overlap. Importantly, this will also enable creation of a molecular atlas of memory B cells and plasma cells across tissues, integrating transcriptomic, phenotypic and B cell receptor repertoire data from the Projects – we will assemble such a data set with an eye to future incorporation into a Data Commons environment. To develop this Core, we have assembled a strong team with experienced leadership and talented individuals with demonstrated expertise in all of the areas covered. Combining these three broad areas into a Core will maximize efficiency, standardize process and promote scientific synergy across the Program.
INFRASTRUCTURE AND OPPORTUNITY FUND MANAGEMENT CORE
LUND, FRANCES E., Core Lead
The NIH Cooperative Centers on Human Immunology (CCHI) program was designed to promote research in human immunology by providing funding for 8-10 Programs as well as additional financial support for pilot projects, new resource and reagent development and/or cross-program collaborative efforts. These developmental awards are made possible through funding provided by the Infrastructure and Opportunity Fund (IOF) grant. The Infrastructure and Opportunity Fund grant is awarded to one of the Programs supported through the CCHI U19 mechanism. The institution that receives the Infrastructure and Opportunity Funds grant is responsible for establishing an Infrastructure and Opportunity Fund Management Core (IOFMC). The IOFMC works with the NIH Program officers and the CCHI scientific steering committee to solicit, review and distribute Infrastructure and Opportunity Funds as subawards to researchers at the IOFMC parent institution and investigators working at other institutions, who are, in many cases, members of one of the other funded CCHI cooperative centers. The IOFMC is responsible for administrative oversight of the subawards and for ensuring that the recipient of the award and the institution in which they work are in compliance with all applicable state, NIH and federal regulations and that charges to the subaward are reasonable and allowable. Finally, the IOFMC must also provide annual financial reports on the awarded subcontracts to NIH. The major goal of the UAB IOFMC will be to establish an administrative structure that will manage the subaward program from the request for applications (RFA) stage, to the pre- and post-award process, and through the grant close- out and reporting phase. In order to meet this goal, we will: (i) coordinate the IOF project application and selection process; (ii) establish subcontracts to distribute IOF development and pilot project grants to Project Leaders at other institutions; (iii) provide administrative and fiscal oversight of these subawards; and (iv) serve as the communications interface between IOF awardees, the CCHI steering committee and the NIH. Successful and timely completion of these objectives by the UAB IOFMC will allow the NIH to leverage the intellectual, infrastructure and unique reagents/samples that are present in the CCHI network institutions to advance the overall scientific goals of the CCHI program.
DEVELOPMENT AND MAINTENANCE OF HUMAN GLYCAN AND PHOSPHOLIPID ANTIBODY REPERTOIRES
KEARNEY, JOHN FRANKLIN, Project Lead
King, Rodney G, Co-Project Lead
Development and maintenance of human glycan and phospholipid antibody repertoires Project Summary Natural antibodies (nAbs) exist in the blood of multiple mammalian species in the absence of deliberate immunization. Reactivity of nAbs with epitopes conserved between pathogens and autologous host antigens allow these antibodies to perform dual functions in immunity: providing important host defense against infection and facilitating housekeeping functions important for tissue homeostasis. The mechanisms controlling human nAb development and maintenance, however, are poorly understood. The goal of this project is to define mechanisms controlling maturation of the human natural B lymphocyte repertoire and its tissue distribution. We will achieve this goal by completing a targeted analysis of B cells reactive with conserved carbohydrate and phospholipid T lymphocyte-independent antigens associated with clinically relevant bacteria and xenoantigens. Through this approach, we will test our central hypothesis that the selection of innate-like B cell clonotypes and antigen-specific tuning of the nAb-producing B cell repertoire depend on interactions with autologous antigens and microbial antigens encountered at mucosal surfaces, which together modulate the entry of B cell clonotypes into the memory and antibody-secreting B cell compartments. In the first Specific Aim, we will sort-purify single, indexed carbohydrate- and phospholipid-binding B cells from a cohort of cadaveric human tissue donors. Immunoglobulin gene expression in these B cells will be analyzed together with expressed cellular phenotype to determine the distribution of clonal networks of nAb-producing B cells across B cell compartments in multiple human tissues. We will additionally examine the stability of innate-like B cell clonotypes and specificity of nAb repertoire by longitudinally sampling human blood after anti-CD20 (rituximab) B cell depletion, to determine the extent of antigen-reactive clonal B cell extirpation and clonal repertoire recovery during B cell compartment regeneration. Specific Aim 2 will utilize a novel, high-throughput antibody-cloning and expression platform to express immunoglobulin gene rearrangements from gene amplicon libraries as recombinant Abs and examine their binding properties, including antigen affinity and fine specificity. We will additionally examine the effects of somatic mutation on the binding properties of nAb by assessing the global reactivity of cloned antibodies and germline-reverted clonotypes using mammalian glycan antigen microarrays. This targeted analysis of antigen- reactive human B cells will permit analysis of fine antigen-specificity, affinity, and avidity of B cell clonotypes across human tissues, and the determination of whether these features of the BCR influence the tissue, B cell subset, and immunoglobulin isotype distribution of certain clones. Because nAb antigens are expressed by multiple commensal and pathogenic organisms and are protective in normal immune homeostasis, such findings will facilitate the development of immunotherapies designed to intersect the natural repertoire.
CHARACTERIZATION OF VIRUS-SPECIFIC HUMAN B CELL SUBSETS IN LYMPHOID AND NON-LYMPHOID TISSUES
LUND, FRANCES E., Project Lead
Randall, Troy D., Co-Project Lead
Characterization of Virus-Specific Human B Cell Subsets in Lymphoid and Non-Lymphoid Tissues Project Summary Antibodies against influenza virus protect against infection and reduce morbidity and mortality. As a result, vaccines against influenza are designed to elicit antibodies against circulating strains of virus. Given that antibodies are made by B cells, it makes sense to characterize influenza-specific B cells in order to determine how they are selected, how they function and where they are maintained. However, most studies of influenza- specific B cells in humans are limited to B cells from peripheral blood, rather than those in lymphoid organs or the lung. Moreover, most studies characterize influenza-specific antibody-secreting cells (ASCs), which circulate for a short period after infection or vaccination. Consequently, we have minimal understanding of influenza-specific memory B cells or ASCs in human tissues. Thus, there is an urgent need to characterize the phenotypes and functions of influenza-specific B cells in human lymphoid and non-lymphoid tissues. Given this gap in knowledge, we are proposing experiments that will use “B cell tetramers” to identify influenza-specific B cells responding to hemagglutinin (HA), nucleoprotein (NP) and non-structural-1 (NS1) proteins of influenza virus in blood, lymphoid tissues, lung and visceral adipose tissues of normal human donors. We will then determine how the phenotypes, transcriptional programs, functional properties, reactivities and affinities of those B cells differ between tissues and between B cells of different specificities. Importantly, we will use an automated, single-cell cloning and antibody-expression system to rapidly and efficiently generate recombinant, influenza-specific antibodies from sorted memory B cells in order to determine their affinity and cross-reactivity. Therefore, we believe that this project is highly innovative and will significantly advance our understanding of influenza-specific B cell biology in various human tissues.
DEVELOPMENT OF NOVEL REAGENTS TO IDENTIFY XENOGRAFT REACTIVE B CELLS
TECTOR, A JOSEPH, Project Lead
Development of Novel Reagents to Identify Xenograft Reactive B Cells Project Summary A shortage of available donor organs is the most critical challenge facing organ transplantation today. Pigs are considered a promising source of replacement tissues. Unfortunately, xenotransplantation, the sharing of organs across species, is not clinically applied because humans exert a strong humoral immune response toward pig tissues. Our long-term goal has been to make pig tissues suitable for use in humans by preventing the binding of human antibodies. The central hypothesis of this project is that pig Major Histocompatibility Complex (MHC) proteins, known as Swine Leukocyte Antigens (SLA) in the pig, contribute to xenoantigenicity. We have shown that patients having antibodies against Human Leukocyte Antigens (HLA) often cross-react with the homologous SLA. We have also found that approximately 25% of the population who lack HLA antibodies also have IgG and IgM capable of binding SLA. Inactivating SLA to eliminate their contribution to human anti-pig immunity is problematic because the SLA are key to helping protect the organ from infection and cancer. The objectives of this grant are to: (i) improve our understanding of the frequency with which patients have anti-SLA antibodies; (ii) determine the origin of B cells which produce those antibodies, and (iii) define their susceptibility to a B-cell depleting therapy (Rituximab). Our approach is innovative because it will develop novel tools that build on similar approaches that have been successful in the setting of allotransplantation. Our rationale is that this knowledge will bring us bring us closer to the use of pigs as an organ source by helping to better match donors with recipients. In Specific Aim 1 we will create recombinant SLA, produce a bead array of these proteins, and use the array to screen 500 human sera for the presence of SLA antibodies. The focus of Specific Aim 2.1 is to screen 25 patients before and after Rituximab treatment with the SLA bead array to determine if those xenoreactive antibodies diminish. Specific Aim 2.2 will identify and characterize SLA-specific B cells to determine if natural antibodies account for some of the SLA reactivity. This technology development will contribute significantly to the ability to match pig donors with human recipients by avoiding or eliminating SLA-specific antibodies. This project will rely on several components of this award to achieve its goals. Core C will provide bead arrays for serum screening and SLA tetramers with which to analyze B cells provided by Core B. Project 2 investigators and Core D will help with the sorting and analyses of tetramer-stained B cells. Project 1 will provide Rituximab samples and data and assist in comparisons to their work to determine if B cell depletion affects SLA-specific antibodies and naturally occurring anti-glycan antibodies similarly.