Human memory B and T cells (Bmem, Tmem) can be found in circulation and residing in lymphoid and non- lymphoid tissues. After infection and vaccination, we can follow antigen (Ag)-specific phenotypically-defined memory cells in blood, but we can’t easily track Ag-specific clones of memory cells in blood over time and the cells are lost to us if they leave circulation and move into tissues. Our knowledge of the antigen (Ag)-specific Tmem and Bmem compartments in tissues, particularly non-lymphoid tissues, has been largely limited to single timepoint analyses. While we have learned much about the human immune system from these static and serial approaches, at the end of the day, these analyses still only provide us with “immune snapshots” of the Ag-specific memory clones and tissue resident memory cells. This limits us from asking key questions such as which signals facilitate or prevent the recruitment, retention, and survival of specific populations of tissue resident human memory cells. We are also unable to precisely measure the evolution of Ag-specific clones over time or assess whether microenvironment or immune perturbations influences the evolution and durability of these clones of cells. This knowledge gap is the basis for the Cooperative Centers in Human Immunology (CCHI) U19 application entitled “Evolution and Durability of Human T and B cell Responses”. The goal of this Program is to address the knowledge gap by leveraging our access to samples from organ transplant recipients to: (i) monitor cellular trafficking of memory cells from circulation into tissues over time; (ii) study the evolution of memory cells that reside in the tissue or respond to tissue specific Ags; and (iii) assess the durability of these memory populations over time and in response to changes in the environment. The central hypothesis addressed in this program is that the human immune memory compartment is constantly evolving in response to local and systemic signals that influence both the durability and reactivity profile of these cells. We will address this hypothesis in three research Projects that are supported by three scientific service Cores. Project 1 will address the factors that control the evolution and durability of memory B cells and antibody secreting cells that contribute to organ failure and rejection in kidney transplant patients. Project 2 will study the generation and durability of memory T cells that reside in the endometrium of uterus transplant patients. Project 3 will assess B cell and antibody responses in lung transplants. The three science service Cores will support the overarching goals of the Program and will provide the individual projects with the tools needed to track and analyze memory T cells, memory B cells and antibodies. We believe that the studies in this Proposal are important because they are designed to address fundamental questions about the generation, evolution and durability of human memory B and T cells. In the future, these data may be used to develop immune-modulating modalities that can be used to appropriately tune protective and damaging memory cell-driven immune responses.
If we want to understand how cells of the immune system, like B cells and T cells, can both protect from infections and cause damage in settings like organ transplant or autoimmunity, it is important to be able to follow the protective and damaging cells over time to see where they go, what they do and how long they live. The goal of this Program is to analyze the different types of B and T cells in samples of blood and biopsies from transplanted tissues that are collected multiple times over months to years from the same transplant patients. In the future, this information could be used to develop better treatments to eliminate the immune cells that cause damage in the organ without affecting the cells that protect us from infections.
Our U19 Center consists of five Cores and three Projects:
CORE A: ADMINISTRATIVE CORE
LUND, FRANCES E., Core Lead
Our understanding of how human adaptive immune responses are initiated, evolve over time, and become durable have been shaped largely, although not exclusively, by what we have learned from measuring B and T cells in the blood following immune perturbations with pathogens, vaccines and immune-modulating drugs. While longitudinal analyses can be performed with blood samples, it is only possible to assess the recirculating immune cells and we know that many antigen-experienced “memory” B and T cells rarely recirculate and reside in peripheral and lymphoid tissues. Recent studies in animals reveal that immune memory cells in tissues play important early roles in immune protection at barrier sites. The generation and maintenance of these cells are controlled by different mechanisms and these tissue-residing memory cells are endowed with distinct phenotypic, molecular, and functional programs. Although it is difficult to study human tissue-residing immune cells, the elegant studies that have been done confirm that studying immune cells in tissues reveals additional complexity that is not always captured in peripheral blood. However, to date, most human studies in peripheral tissues have not evaluated the evolution or durability of antigen specific B and T cell responses in the face of a continuously changing microenvironment and/or ongoing antigen exposure. This gap in knowledge limits our ability to identify appropriate modalities to boost and maintain protective immune cells within peripheral tissues and to prevent the development and persistence of potentially pathogenic immune cells that contribute to tissue damage. This U19 addresses the knowledge gap by characterizing immune memory cells that reside in tissue samples derived from transplant patients. The unifying goals of the three U19 projects are to better understand the evolution of human immune memory responses in tissue and blood and to characterize the factors that contribute to the persistence and durability of human memory T and B cells. To meet these goals, investigators with complementary expertise in B and T cell biology, infectious disease, transplantation and autoimmunity will work together to methodically interrogate the memory B and T cell compartments in transplanted kidney, uterus and lung. The major objective of Core A is to provide financial, scientific and regulatory oversight and administrative support for the overall Program as well as the individual projects and cores. We will achieve this objective by: (i) supporting the scientific progress of the projects and cores, organizing the monthly research in progress meetings and interfacing with the project and core team members to ensure needs are met; (ii) organizing scientific interactions such as the annual retreat with the scientific advisory board, and the annual Cooperative Centers on Human Immunology investigator meeting; (iii) providing financial oversight and administrative support to the Program; and (iv) organizing the keynote lecturer at the annual UAB Immunology Institute Vaccine Symposium. The Administrative Core team members will ensure that the components operate as a synergistic, cohesive unit.
CORE B: ANTIGEN RECEPTOR SEQUENCING, CLONING, EXPRESSION and ANALYSIS CORE
KING, RODNEY G., Core Lead
The B cell compartment is comprised of many individual cells, each of which may express a distinct B cell receptor (BCR). Upon antigen engagement, B cells proliferate resulting in clonal expansion, and may further diversify their BCR genes through somatic hypermutation (SHM) and class switch recombination. These two processes allow for affinity maturation toward the insulting antigen and the distribution of Ab reactivity across isotypes which interface with other components of the immune system defining an Abs effector function. Clonal expansion and diversification result in the presence of expanded groups of B cells with similar BCRs (clonal lineages). The overarching goal of this program is to investigate the evolution and durability of immune responses to allo, self, and foreign antigens. Although sequencing of BCR genes allows the visualization of cell expansions and diversification that occur during the evolution of B cell responses, sequence information alone is insufficient to evaluate changes in antigen binding that occur as immune responses evolve. The Antigen Receptor Sequencing, Cloning, Expression, and Analysis Core (Core B) will facilitate the objectives of this program by allowing the analysis of these two distinct but related features of humeral immunity. We will support the aims of the projects through the following activities: i) Generating antigen receptor sequence information. Projects require repertoire level sequence information form bulk and sorted lymphocytes, BCR for Projects 1 and 3, TCR for Project 2. Core B will centralize the processing and quality control involved in generating next-generation of antigen receptor libraries. We will coordinate with the Projects and with Core D, the Data and Informatics Service Core, for analysis. ii) Generating Abs by cloning and expressing the BCR of single-sorted antigen-reactive B cells as recombinant Ab. Both Projects 1 and 3 of this proposal are evaluating the reactivity of antigen-reactive B cells to a variety of specificities, allo- and auto-HLA, and viral pathogen derived antigens. Using a high- throughput Ab cloning and expression system, Core B will produce recombinant Abs from the cloned BCR of single-sorted antigen reactive B cells across reactive with these antigens. iii) Evaluating the specificity, relative affinity, and cross reactivity of Abs. Because understanding the effects of SHM, the relation of antigen reactivity within and across expanded lineages, the relative affinity of Abs to their antigens and related structures requires physical measurements of binding, Core B will evaluate Abs reactivity profiles using multiplexed cytometric arrays. iv) Core B will generate Ab and Fab expression vectors to support the bio-physical measurements of Ab binding and structural studies performed by Core C. Collectively, these activities will promote synergy between the Projects and the Cores of this proposal and accelerate the research by providing access to specialized techniques and reagents while centralizing approaches requiring technical expertise and instrumentation.
CORE C: IMMUNO-REAGENT PRODUCTION, VALIDATION and BIOPHYSICAL ANALYSIS CORE
GREEN, TODD J., Core Lead
Patients receiving organ transplants have the risk of organ rejection due to mismatched human leukocyte antigens (HLA). Though organ recipients are screened for pre-formed anti-HLA antibodies (HLA-Abs) and are cross-matched using donor cells and recipient serum prior to transplantation, up to 25% of kidney transplant recipients will develop a new donor-specific HLA-Ab following transplant. Within that patient group, there is a 40% decline in 10-year graft survival. At the heart of this negative outcome are antibody-secreting cells (ASCs), which produce donor-specific Abs, and memory B cells (Bmems), which may provide help to alloreactive T cells and differentiate into ASCs. Current immunosuppressive therapies aimed at depleting ASCs and Bmems are largely ineffective. Thus, Ab-mediated rejection remains a significant clinical problem. The overall objective of this U19 Program is to define the factors that govern the evolution of memory lymphocyte responses over time and to assess the durability of these responses. Two Projects in this U19 will specifically focus on characterizing the evolution and durability of the Bmem and ASC subsets responsible for alloimmune responses elicited toward donor-specific HLA Class-I and Class-II alleles. To perform these studies, Projects will require antigen-specific reagents to aide in identification of antigen-specific B cells, clone B cell receptors, and further characterize antigen to Ab interactions. One objective of the Immuno-Reagent Production, Validation, and Biophysical Analysis Core (Core C) is to provide these reagents, which will include B cell tetramers (specific to HLA alleles and respiratory viral antigens), Abs, Fabs, antigen-specific polyclonal antibodies (pAbs), and proteins for structural studies. By centralizing the production and validation of proteins for distribution within the Program, the expertise of Core C will allow Projects to focus on their own Aims, unimpeded by the time and effort to develop these reagents on their own. A second objective of Core C is to function as a multidisciplinary biophysical analysis center. In this capacity, we will coordinate with Projects to analyze antibody to antigen interactions. Using surface plasmon resonance, we will develop an understanding of these interactions at the kinetic level. Using X-ray crystallography and cryo-electron microscopy, we will observe epitope-paratope interfaces at near atomic resolution. Lastly, we will perform proteomics experiments using mass spectrometry with pAbs to aide in assigning pools of Abs to lineages. In summary, Core C will provide reagents to the exceptional group of Immunologists in the Program to expedite their discoveries. Biophysical analyses will provide new innovation and synergy to these groups. Structural biology will be a transformative tool for the Projects that will lead to assignment of rules regarding HLA engagement by alloAbs. Filling this knowledge gap could lead to improved donor-to-recipient HLA matching protocols and lower alloreactive Ab responses, leading to better long-term outcomes in transplant patients.
CORE D: DATA and INFORMATICS SERVICE CORE
ROSENBERG, ALEXANDER, Core Lead
Hypothesis-testing studies designed to understand the evolution and durability of B and T cell responses in the contexts of alloreactivity, tissue residency and viral infection that are described in this proposal will rely on several `omics and other data-rich approaches and corresponding analyses. To support use of these platforms and their associated data analyses, we propose to provide unified and integrated data and informatics services in the Data and Informatics Service Core (Core D). The core will cover two broad areas as reflected by the Specific Aims: data management, including clinical data integration, and bioinformatic and statistical data analysis, both primary (standardized pipelines) and downstream. In Specific Aim 1, we propose to oversee and implement data management processes and systems applied to both raw and derived data as well as its integration with corresponding clinical characteristics of human donors. These efforts will support raw-to-figure analytical provenance and 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, support 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 and data generated by one or more Cores), including bulk B and T cell receptor repertoire sequencing, single-cell sequencing applications (gene expression, VDJ, feature barcoding for surface phenotyping and antigen specificity, spatial transcriptomics, multiome), analysis of antibody reactivity profiles and other assays. This will benefit the Program by standardizing primary data processing across the Projects and will promote comparability of results. Additionally, 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. 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 two broad areas into a Core will maximize efficiency, standardize processes, and promote scientific synergy across the Program.
INFRASTRUCTURE AND OPPORTUNITY FUND MANAGEMENT CORE (IOFMC)
LUND, FRANCES E., Core Lead
The NIH Cooperative Centers on Human Immunology (CCHI) are designed to promote human immunology research focused on understanding the mechanisms underlying human immune system regulation and function; the ultimate goal being the prevention and treatment of infectious and immune-mediated diseases. The Program provides funding for 6-9 Centers as well as additional financial support for collaborative and pilot/feasibility projects, resource and reagent development, translational projects, and/or early-stage investigator projects provided through the Infrastructure and Opportunity Fund (IOF). One of the Centers funded through the CCHI U19 mechanism is selected to establish an Infrastructure and Opportunity Fund Management Core (IOFMC), which bears the responsibility of managing and distributing the IOF. Working with the NIH Program Officer and the CCHI Steering Committee, the major goal of the UAB IOFMC will be to establish an administrative structure that will manage the supplemental projects from the solicitation of applications, through pre- and post-award, to award close-out and reporting. The UAB IOFMC will: i) coordinate the IOF project solicitation, application and selection process; ii) establish subcontracts to distribute IOF supplemental awards to recipients at other institutions, usually another CCHI Center, but often a collaborating institution; iii) provide administrative, reporting and fiscal oversight of the IOF awards to ensure that the recipient of the award and the institution in which they work are in compliance with all applicable NIH and federal regulations and that charges to the subaward are reasonable, allocable and allowable; iv) support the NIH Program Officer and CCHI Steering Committee in the execution and advancement of programmatic decisions; and v) serve as the liaison between the IOF awardees, CCHI Steering Committee and the NIH. The IOFMC will also develop and manage a website for CCHI activities. Completion of these objectives by the UAB IOFMC will allow the NIH to successfully leverage the existing and future intellectual, infrastructure and unique reagents/samples available within the CCHI program to advance the overall scientific goals of the CCHI.
EVOLUTION and DURABILITY of ALLO(AUTO)IMMUNE B CELL RESPONSES in ORGAN TRANSPLANT RECIPIENTS
LUND, FRANCES E., Project Lead
Antibodies (Abs), which are produced by B lineage-derived Ab secreting cells (ASCs) following pathogen exposure and vaccination, play critical roles in protection from infection. However, ASCs and Abs can be pathogenic if directed against the wrong target, as is the case for autoimmune disease and organ transplantation. In the setting of kidney transplant (KT), Abs made by the recipient directed against the donor’s Major Histocompatibility Complex or Human Leukocyte Antigen (HLA) proteins (donor-specific Ab, DSA) contribute to Ab-mediated rejection (AMR). Pre-formed DSA at the time of transplant can cause acute AMR and de novo DSA, which is elicited months to years after transplantation, can cause chronic AMR. Both conditions are difficult to treat and associated with organ failure. Thus, while overall transplant success rates have improved, there is still a need to better understand the fundamental mechanisms that control the development, evolution and durability of the B cell and B cell-derived DSA responses against donor (allo) HLA Class-I and Class-II. In fact, we know remarkably little about B cell responses to alloHLA proteins, which unlike pathogen-derived antigens, are very similar to the HLA proteins expressed by the patient (selfHLA). Studies using Rituximab to deplete circulating B cells in DSA+ transplant patients reveal that DSA levels remain high in many patients. These data support the idea that the DSA response is largely driven by Rituximab-resistant long-lived bone marrow ASCs (LL-ASC). However, memory B cells (Bmem), particularly those that reside in tissues, are also more resistant to Rituximab and both Bmem and LL-ASCs are formed by germinal center (GC) B cell responses that can take place in lymphoid and inflamed tissues. Bmem, which can rapidly differentiate into new cohorts of ASCs, could potentially serve as a reservoir to sustain systemic Ab responses. We found that HLA-reactive Bmem were present in the blood and kidney of a DSA+ KT patient diagnosed with AMR. These HLA-specific B cells were present in expanded B cell lineages and the BCRs expressed by these B cells were high affinity, class-switched and extensively somatically mutated, suggesting that they were generated via a TFH-dependent GC response. Moreover, we observed extensive clonal relationships between the Bmem and ASCs in both tissues and we identified Bmem and ASCs lineages that contributed to the serum DSA response. Intriguingly, many of the recombinant monoclonal Abs derived from multiple HLA-reactive B cell lineages bound both donor alloHLA and selfHLA. Therefore, we hypothesize that ongoing evolution of the alloHLA B cell response occurs in the face of a durable low affinity selfHLA response, which is not censored. The immediate goals of Project 1 are to: (i) define the B cell populations in blood and kidney that contribute to HLA reactivity, and (ii) determine how the kidney and recirculating alloHLA reactive B cells evolve with time and following immune perturbations. We believe these experiments are important as gaining a more complete understanding of how B cell responses to alloHLA are initiated and maintained may, in the future, allow for better design of therapies to prevent or treat AMR.
GENESIS and DYNAMICS of HUMAN ENDOMETRIAL RESIDENT T CELLS REVEALED BY UTERUS TRANSPLANT RECIPIENTS
PORRETT, PAIGE M., Project Lead
Most T cells in the human body are located within individual tissues as antigen-experienced, resident memory T cells (TRM). However, much of the scientific knowledge about human T cells has been generated from studies of peripheral blood and secondary lymphoid organs where most T cells are naive. Unfortunately, many of the lessons learned from T cells in peripheral blood may therefore not apply to tissue resident immune cells because tissue environments shape immune cell differentiation and responses in ways that do not occur in other anatomic compartments. We therefore lack the knowledge necessary to manipulate tissue resident cells to our advantage to treat a variety of diseases and patient populations. Tissue resident memory T cells are an antigen-experienced, adaptive immune population that play a critical role in protective immunity to pathogens. We address this knowledge gap in this proposal through the study of TRM in the human uterine endometrium. Notably, TRM recruitment, composition, and longevity may be distinctly different in the human endometrium versus other tissues because the endometrium undergoes hundreds of cycles of coordinated tissue loss and regeneration over the course of a lifetime. We have combined cutting-edge next generation sequencing methodologies with access to the endometrium of healthy control volunteers and human uterus transplant recipients to answer questions about TRM trafficking and differentiation that will advance our understanding of human tissue immunity. We expect that this proposal will have particular impact for women’s health and transplant recipients but may also inform vaccine strategies and cancer therapeutics as well. In summary, we expect that the knowledge generated by this proposal can be used to expand our capabilities to modulate the human immune system and treat a variety of human diseases.
EVOLUTION, DYNAMICS and DURABILITY of B CELL AND ANTIBODY RESPONSES in LUNG TRANSPLANTATION
RANDALL TROY D., Project Lead
Emerging data suggest that, like lung-resident memory T cells, memory B cells can reside in the lung without recirculating and act as first responders to pulmonary pathogens like influenza and SARS-CoV-2. We termed these cells lung-resident memory B cells or BRM cells. We also showed that lung-resident memory B cells require contact with antigen within the lung in order to initiate their residency program. Taken together, these data suggest that BRM cells are an important component of immunity to pulmonary pathogens. However we have only a rudimentary understanding of where BRM cells come from, how they are selected, what antigens they react with and how they are recalled (or not) after vaccination or secondary infection. Although lung-resident memory B cells are clearly generated in response to pulmonary infection, it is less clear whether they are generated in response to other types of antigens like auto-antigens or allo-antigens. Interestingly, many pulmonary diseases, like COPD, IPF and ILD, have an autoimmune component in some patients, perhaps as a consequence of persistent inflammation. Moreover, allo-reactive antibodies are often observed in lung transplant patients. Given that auto-antigens and allo-antigens are widely expressed in the lung tissue, it makes sense that lung-resident B cells will respond to these antigens in the lung. Our overall hypothesis is that allo-reactive, auto- reactive and pathogen-reactive B cells in lung allografts are primed and selected locally in the lung, and that the signals and cellular interactions involved in this process are different than those in conventional secondary lymphoid organs. To test this hypothesis, we will take advantage of single cell methods that allow us to define (and compare) individual B cells in blood, bronchalveolar lavage (BAL) fluid and lung tissue by a combination of transcriptome (single cell RNseq), BCR clonotype (single cell BCRseq), DNA-barcoded antibodies to surface markers (CITEseq) and affinity/specificity/cross-reactivity of BCRs cloned and expressed as recombinant antibodies (single cell cloning). We will also use high-dimensional antigen arrays of HLA alleles, auto-antigens, and virus-derived antigens to quantify the reactivity of antibodies in the blood and BAL fluid of lung transplant patients. Using these methods to compare populations of auto-reactive, allo-reactive and virus-reactive-specific B cells in the lung, BAL and blood over time and, in some cases, after infection, we will be able to determine how memory B cells in the lung are related to one another, the depth of their selection, the extent of their cross- reactivity and their ability to respond to local antigens. This information will be informative about the evolution of donor-specific antibodies in the context of lung transplant, the potential role of auto-antibodies in the lungs of transplant patients and the ability of pre-formed, virus-reactive (DONOR) B cells retained in donor lungs as they respond to infection.
