Gabriel Victora, PhD from the Rockefeller University. Project is entitled “Reactivation and secondary hypermutation of memory B in humans following hepatitis B immunization”
A feature of the immune system of critical importance is its ability to mount a much stronger antibody response the second time a pathogen or antigen is encountered. This property underlies the need for “booster” doses to ensure the effectiveness of vaccination. The booster effect derives in part from the generation, by the primary response, of expanded clones of memory B cells (MBCs). Upon re-exposure to antigen, MBCs rapidly proliferate and differentiate into plasma cells, generating high titers of serum antibody over a short period of time. Because most MBCs that respond to boosting have also undergone affinity maturation in germinal centers (GCs) during the primary response, MBC-derived antibody has both higher affinity and higher cross-variant breadth than primary antibody. A common assumption in the vaccinology field has been that, in addition to forming plasma cells, MBCs will also generate secondary GCs upon boosting with high efficiency, allowing them to re-evolve their immunoglobulins to adapt to variant strains of a pathogen. This assumption forms the basis of multiple attempts to guide B cell clones towards broad reactivity to influenza or HIV by iteratively recruiting them to germinal centers by sequential immunization. Long histories of repeated exposure can generate influenza-specific MBC compartments in blood that represent >1% of all B cells. On the other hand, brief exposure regimens, such those typical of HPV, tetanus, and HBV vaccination, generate much lower frequencies of antigen-specific MBCs (in the high tens to low hundreds per million B cells), which are comparable to frequencies found after single immunization or infection in mice. We therefore hypothesize that the wide range of MBC re-entry into recall GCs observed after human influenza vaccination reflects differences in the history of exposure of individuals to influenza antigens, rather than more fundamental differences in MBC biology between humans and mice. Our primary aim is to determine the exact contribution of germinal center-derived MBCs to secondary GCs under conditions of known and controlled exposure. We expect the findings of this study will be of critical value to our general understanding of vaccination, as well as to those seeking to generate influenza and HIV broadly neutralizing antibodies (bNAbs) by vaccination. Our studies will also be critical to determine the extent to which mouse models reliably predict human responses at the clonal level and can therefore be used to test sequential immunization regimens.
Debattama Sen, PhD from Massachusetts General Hospital. Project is entitled “Dissecting exhausted CD8+ T cell fate and function using epigenome editing”
The functional impairment of T cell-mediated immunity is a defining feature of chronic viral infections such as Hepatitis C and HIV, as well as many cancers. Indeed, CD8⁺ T cell dysfunction is now recognized as a critical determinant of tumor progression and resistance to immunotherapy regimens which harness the immune system directly to fight cancer. Exhausted T cell portray a variety of functional defects including impairment of survival and proliferative capacity, loss of effector cytokine production, and diminished cytotoxicity. T cell exhaustion in humans is associated with a unique transcriptional signature compared to other contexts of T cell dysfunction like anergy. To delve deeper into mechanisms of gene regulation in chronic settings, we have performed ATAC-seq in exhausted HCV- and HIV-specific T cells to profile the landscape of accessible chromatin, which is enriched for regulatory elements such as active enhancers. These studies revealed for the first time that exhausted cells acquire an extensive, state-specific epigenetic program that is distinct from memory T cells.
Recent work from the Sen and Lauer labs in chronic HCV infection suggests that the epigenetic program of T cell exhaustion, once established, is not fully reversed by direct-acting antiviral (DAA) treatment that eliminates chronic viremia. Instead, functional defects remain in human CD8⁺ T cells despite cure of chronic infection. This phenomenon impairs protective memory formation after reduction/elimination of viral loads in chronic HIV and HCV infection. Furthermore, these epigenetic and molecular “scars” are likely to occur in cancer where they may impact the effectiveness of engineered T cell therapies. Indeed, in both solid and hematologic malignancies, dysfunction of transferred T cell therapies and lack of persistence remains a major problem. Reinvigorating effector function in chronic infection and cancer will require directly targeting the altered epigenetic regulation in exhausted CD8⁺ T cells. Here, we have taken two orthogonal strategies of targeting exhaustion-associated transcription factors (TFs) as well enhancers that cannot be reversed by cure of infection. Specifically, we focus on the regulation of key effector functions in CD8⁺ T cells including the production of key cytokines such as IFN-γ, expression of inhibitory receptors, and capacity to proliferate upon antigen (Ag) stimulation. We propose the following specific aims:
Aim 1: Edit state-specific enhancers governing key effector molecules in functional vs. exhausted CD8⁺ T cells. In this aim, we will use CRISPR/Cas9 genome editing of endogenous enhancers to identify how IFN-ψ production is differentially regulated in Flu-, EBV-, CMV- and HCV-specific CD8+ T cells.
Aim 2: Perturb transcription factor activity to improve antiviral immunity in human CD8⁺ T cells. In this aim, we will examine whether loss of exhaustion-associated TFs (TOX, EOMES, ETV1, NKX.3) can improve effector cytokine production and proliferative capacity in functional vs. exhausted CD8⁺ T cells.
Ansuman Satpathy, MD, PhD from Stanford University. Project is entitled “Massively-parallel single-cell multi-omics to chart human immune cell states in infection”
Although every cell in the body contains the same genome, cell type- and cell state-specific gene regulation programs establish gene expression necessary for cellular specialization. This bridge from identical genotype to disparate phenotype is coordinated by a complex interplay of millions of cis– acting DNA elements and thousands of trans-acting transcription factors. A striking observation from epigenomic studies is that RNA expression only provides a partial view of a cell’s underlying gene regulatory networks, and that epigenomic profiling can identify a complementary view of cell state-specific regulation that controls cellular development and function (Corces et al, Nat Genet, 2016). This view of gene regulation is particularly important for understanding the molecular impact of disease-associated genetic risk variants, which reside in non-coding DNA regions in up to ~90% of cases (Farh et al, Nature, 2015). However, the study of gene regulation in human immunology has been limited by previous protocols for genomic profiling, which required large numbers of cells (upwards of 100 million), and thus precluded their application to human specimens and rare immune cells in these samples. These methods also generally measured a single biomolecule class, and therefore, scientific questions that require multi-modal data have been intractable.
Here, we propose a new assay termed Scale-ASAP-seq (Scalable ATAC with select antigen profiling by sequencing), an innovative new assay ideally suited for examining the molecular programming and clonal lineage of millions of human immune cells in routine experiments. In each single cell, our proposed assay would yield: 1) a genome-wide epigenetic landscape of chromatin accessibility; 2) the abundance of >270 proteins, both intracellular and cell surface; 3) inference of clonal lineages via measurement of somatic mitochondrial DNA (mtDNA) mutations, and 4) CRISPR sgRNA sequences, if desired. After establishing this single-cell technology, we hope to utilize our multi-omic approach to interrogate immune cells at an unprecedented resolution and throughput in the context of human infection, and in collaborations with the CCHI consortium and investigators.
Anamika Patel, PhD from Emory University. Project is entitled “Structure-Function study of CoV-2 neutralizing antibodies from Indian population”
Since its emergence in 2019, SARS-CoV-2 has quickly spread throughout the globe, with the USA and India encountering the highest number of cases and over a million deaths. Additionally, new variants continued to emerge worldwide. India experienced the most devastating consequences when the delta variant first emerged, which then rapidly spread globally. It is plausible that some of the new variants are less likely to be neutralized by antibody defenses induced by previous natural infections or vaccines. Therefore, there has been a paramount interest in understanding the epitope sites targeted by the antibodies to gauge their susceptibility against emerging variants. Accordingly, several elegant studies have taken the approach of making human monoclonal antibodies (MAbs) from the effector or memory B cells derived from COVID-19 recovered individuals and interrogate their binding/neutralizing epitopes and cross reactivity to emerging variant – some of which result in well-defined monoclonal antibody cocktails that can be tailored for therapeutic applications. While many of these studies have focused on samples from people in the United States, Europe, and China, so far, there is very limited information from the Indian subcontinent. A detailed characterization of human monoclonals derived from COVID-19 recovered individuals from India will supplement these efforts further to prepare well for future epidemics, vaccine development, evaluation, implementation and other interventions. Hence, this proposal is based on this rationale. The proposed work is a team collaboration: Dr. Murali-Krishna Kaja, who leads the joint laboratory of ICGEB-EVC, a unique partnership between international center for Genetic Engineering and Biotechnology (ICGEB, New Delhi, India) and the Emory Vaccine Center (EVC); Dr. Anmol Chandele, a young scientist who is stationed full time associate professor at the ICGEB new Delhi to lead the ICGEB-EVC program group; and Dr. Mehul Suthar, who pioneered characterization of neutralizing responses to COVID-19. Our preliminary studies conducted using a limited set of the MAbs raise some important questions on functional and structural aspects of the MAbs, as some MAbs show strong neutralizing effects, and some do not, despite their strong affinity to RBD – an understanding of which is important given the diverse landscape that is likely to be present in the Indian population. Based on these observations, we hypothesize that a detailed study of MAbs derived from COVID-19 recovered individuals from India by characterizing their functional binding, mapping neutralizing epitopes and cross reactivity to older and newly emerging viral strains will complement our efforts to improve understanding of global COVID-19 responses and intervention strategies.
Aim 1. Perform a comprehensive functional analysis of the MAbs generated from the COVID-19 recovered individuals from India.
Aim 2. Gain mechanistic insights into highly potent MAbs generated from aim-1 by epitope mapping.
Selene Meza-Perez, PhD from the University of Alabama at Birmingham. Project is entitled “Role of microbiota-dependent arginine catabolism in tuning mTOR-dependent Treg function”
Tissue-resident regulatory T cells (Tregs) maintain self-tolerance, prevent inflammation and sustain homeostasis in various organs using tissue-specific mechanisms. For instance, visceral adipose tissue (VAT)-Tregs maintain adipose tissue homeostasis and metabolism using a unique adipose-specific program. VAT-Tregs exhibit several features distinct from their lymphoid tissue counterparts, such as high expression of the transcription factor PPARg, the surface marker ST2 (a component of the IL-33R) and molecules involved in lipid metabolism (CD36). In addition, VAT Tregs possess a restricted and clonally expanded T cell receptor (TCR) repertoire, which suggests specificity to local peptides. Importantly, Treg activation, differentiation and development are dependent on metabolic functions controlled, in part, by the mammalian target of rapamycin (mTOR). Among other metabolites, cellular amino acid uptake of arginine and leucine promote mTORC1 activity in Tregs, whereas its deprivation abrogates Treg suppressive function [17, 18]. Thus, mechanisms that influence mTOR activity in Tregs regulate their metabolism and suppressive function and thereby influence the balance between tissue homeostasis and inflammation.
Gut microbiota composition also significantly impacts numerous aspects of host physiology, since metabolites produced (or consumed) by the microbiota can induce, enhance, and modulate immune cell functions in a variety of tissues, whereas the lack of microbiota alters host nutrient quantities. In general, high taxa diversity is considered a healthy microbiota in humans. Commonly, the phyla Firmicutes and Bacteroidetes dominate the gut microbial community, while members of Proteobacteria, Actinobacteria and Verrucomicrobia are less abundant. Imbalance in the taxonomic composition of the microbiota, called dysbiosis, is well documented in several metabolic disorders. For example, high abundance of Proteobacteria with respect of Bacteroides is commonly associated with risk of metabolic disorders and inflammation. Interestingly, Proteobacteria are enriched in phosphagen kinases, enzymes that utilize arginine as substrate to obtain energy from ATP. As a result, dysbiosis that alters the proportion of Proteobacteria may generate an imbalance in host nutrients like arginine that could affect immune cell functions. At present however, there is a gap in knowledge that mechanistically links specific bacterium with metabolites that influence adipose tissue homeostasis.
In this regard, we have identified in mice that dysbiosis caused by decrease in gram-negative bacteria correlates with increase in arginine levels in serum and high mTOR expression/activity in VAT- Tregs from the omentum, thereby making them dysfunctional. Based on these data, we hypothesize that dysbiosis linked to decreased abundance of Proteobacteria species in humans will correlate with increase in amino acid levels in plasma such as arginine, which will alter Treg metabolism and impair Treg function in adipose tissue. We also hypothesize that these characteristics will be linked to adipose tissue inflammation and metabolic dysfunction. We believe that our experiments will significantly advance human immunology because they will be the first to provide a mechanistic association between Treg function and gut microbiota in the adipose tissue.
Sixto Leal, MD, PhD from the University of Alabama at Birmingham. Project is entitled “The Impact of SARS-CoV-2 Immune Dysregulation on Antifungal Immunity”
Up to 35% of patients in the intensive care unit with severe SARS-CoV-2 (SARS2) infection exhibit positive laboratory testing consistent with 2° mold infection with mortality rates exceeding 40-60%. At UAB, 10% of bronchoalveolar lavage (BAL) fluids from patients with severe viral infection test positive for Aspergillus species including A. fumigatus. Unlike most invasive mold infections, COVID Associated Pulmonary Aspergillosis (CAPA) occurs in individuals with otherwise intact immune systems suggesting novel biological mechanisms mediating susceptibility to fungal infection. Despite hundreds of published articles, meta-analyses, and reviews describing the clinical syndrome of CAPA, there have been zero publications to date exploring the mechanism by which individuals with severe COVID succumb to 2° mold infection. This proposal seeks to evaluate the novel conceptual advancement that immune responses targeting intracellular viral pathogens promote rapid iron-dependent spore germination and compromise essential lower airway and human neutrophil (PMN) antifungal effector functions required to kill large extracellular fungal hyphae. Consistent with the CCHI U19 mission to advance our understanding of human mucosal immune responses to infection, Aims 1-2 seek to explore the hypotheses that necroptosis-dependent pulmonary epithelial cell (PEC) death increases the availability of nutrients and iron resulting in siderophore-dependent increases in spore germination and accelerated fungal growth. Concurrently, SARS2 infection and the antiviral cytokine milieu mitigate the production of PMN-recruiting chemokines resulting in delayed PMN recruitment and spore germination into large invasive hyphae that overwhelm antifungal effector mechanisms resulting in 2° mold infection in an otherwise immunocompetent host. This pilot study is built upon a body of previously published work from our group characterizing the host response to bacterial and fungal infections in human tissues and unpublished preliminary results described below in each specific aim. Successful completion of this pilot study will generate preliminary data to support the submission of a NIH grant application and establish a solid foundation to explore mechanistic hypotheses mediating 2° mold infection in otherwise immunocompetent individuals.
Hypothesis: SARS-CoV-2 mediated pulmonary epithelial cell damage promotes 2° mold infection via release of bioavailable iron, delayed neutrophil recruitment, and decreased antifungal effector expression.
Aim 1: Quantitation of the bioavailable iron, host iron sequestration effectors, neutrophil-recruiting chemokines, and fungi in the lower airway during severe SARS-CoV-2 and CAPA infection. In Aim 1, we will utilize iron detection assays, ELISAs, cytokine protein arrays, PCR, and ITS sequencing to characterize and quantify labile iron, heme, host iron defense proteins, PMN-recruiting chemokines, and fungal pathogens in BAL samples from patients with severe COVID, non-COVID pneumonia, and CAPA.
Aim 2: Characterization of lytic programmed cell death and antifungal effector expression in the lower airway during severe SARS-CoV-2 infection. In Aim 2, we will evaluate autopsy lung tissues from patients that died from severe COVID, non-COVID pneumonia, and healthy lung controls by immunohistochemistry to detect markers of lytic programmed cell death (PCD) and NanoString digital spatial transcriptome profiling to characterize antifungal effector expression within the context of the infected tissue.
William Hildebrand, PhD, from the Oklahoma Medical Research Foundation. Project is entitled “HLA Typing Core for CCHI Investigators”
The major histocompatibility complex (MHC) is a large gene family found in all vertebrates that encodes class I and class II cell surface immune receptors. In humans, MHC molecules are referred to as Human Leukocyte Antigens (HLA) and are subdivided into class I (HLA-A, -B, & -C) and class II (HLA-DR, -DQ, & -DP) molecules. Class I HLA molecules are found on all nucleated cells of the body and they alert the immune system of pathogen-infected and cancerous cells to trigger destruction of the diseased cell by cytotoxic T lymphocytes. Class II HLA molecules are expressed primarily on professional antigen presenting cells including B cells, macrophages, and DC. Unlike the intracellular spaces surveyed by class I molecules, class II HLA molecules are responsible for surveying extracellular spaces for threats such as a bacterial infection. Molecules such as CD1a, c, & d present lipid ligands to cellular immune mechanisms, and MR1 (MHC related protein 1) samples metabolites for T cell review following infection by pathogens such as Mycobacterium tuberculosis (Mtb).
Our Laboratory at the University of Oklahoma Health Sciences Center (OUHSC) pioneered Human Leukocyte Antigen (HLA) typing by DNA sequencing and we continue to provide CLIA (Clinical Laboratory Improvement Amendments)-certified and ASHI (American Society for Histocompatibility and Immunogenetics)-accredited high resolution typing of HLA for clinical transplantation and NIH grantees that study infectious disease and vaccine development. This application seeks U19 Opportunity Funding for our HLA Typing Core following 11,316 typings in five years of U19 funding (April 2015 to March 2019, Mark Davis PI) with 790 typings accomplished through September of the sixth year (December 18, 2019 to April 1, 2021, Frances Lund PI). Thus, in 5¾ years we have completed 12,106 HLA typings for CCHI investigators; the U19 HLA typing core has been steadily utilized. With U19 investigators continuing to send samples for HLA typing, we propose that support for the core is justified to provide typing services to CCHI collaborators and to further advance the HLA typing services offered to these investigators
Sean Brady, PhD from Rockefeller University. Project is entitled “Human-microbial-lectins regulate the mucosal immune system”
It is increasingly understood that the human microbiome is critical to normal immune system physiology and when microbiome-immune interactions are disrupted, it can lead to the development or exacerbation of immune-mediated diseases. Using functional metagenomics screening methods, we identified a number of microbiota genes (i.e., commensal bacteria effector genes, Cbegs) that encode for diverse mechanism by which microbes can perturb human inflammatory pathways. One Cbeg identified in multiple samples (Cbeg5) was predicted to encode for the production of a lectin, however, despite the importance of lectins to human and microbial physiology, there have been no systematic studies of lectins produced by human commensal bacteria (i.e., human-microbial-lectin).
Aim 1. To unravel the impact commensal encoded lectins may have on the human immune system. we propose to recombinantly produce structurally diverse human-microbial-lectins
Aim 2. To screen these proteins against primary immune cells to assess the specific immune response each lectin induces (i.e., immune phenotyping)
We believe that this systematic classification of human-microbial-lectins will lead to an understanding of fundamental mechanisms linking the microbiome to human immunity and facilitate future research into both the role of lectins in immune-mediated diseases and their potential use as therapeutics.
Junyue Cao, PhD from Rockefeller University. Project is entitled “Investigation of the pathogenesis of Psoriasis through a novel single-cell genomic technique”
Psoriasis is a common skin disease defined by epithelial cell hyperplasia and tissue infiltration by activated T-lymphocytes. The interaction between keratinocytes and lymphocytes is the autoimmune basis of this disorder.
For this project, we propose developing a single-cell genomic technique to comprehensively characterize diverse cell types within psoriasis lesion and systematically identify cell-type-specific microenvironment, including the interactions between cells and surrounding cytokines.
This approach will be powerful as transcriptome and chromatin accessibility information of every single cell will be co-profiled with its surrounding environmental signals. With the resulting data, we will have the potential to recover the detailed mechanism underlying abnormal proliferation and differentiation of keratinocytes, and investigate how the activation of T-lymphocytes leads to the disruption of skin homeostasis, and envision that it can be applied to investigate the basis of other autoimmune diseases such as IBD and arthritis.
Einav Shirit, MD from Stanford University. The project is entitled “Unique human lung organoids to study COVID-19 pathogenesis and therapy response”
The pathogenesis of SARS-CoV-2 infection remains poorly characterized, in part due to the limited understanding of the specific cellular targets of this virus; moreover, the cellular and molecular factors that govern differential clinical outcomes across genders, age, ethnicities, etc are unknown. Lastly, while multiple host-targeted approaches of treatment are currently being studied, potential differences in the interpatient susceptibility to these drugs have not been assessed. To address these challenges, we have established a unique PBMC-supplemented lung organoids model derived from normal human tissue.
This project’s main goal is to characterize viral infection, inflammation and tissue injury in this model and use it in proof-of-concept studies to monitor host responses to SARS-CoV-2 infection at high resolution, define the relevant target cells, and monitor response to representative host-targeted approaches.
Our hypothesis is that PBMC-supplemented human lung organoids can capture both interpatient and tissue heterogeneity in the host response to SARS-CoV-2, constituting a unique model to study cellular and molecular determinants in COVID-19 pathogenesis in distinct cell types and patient populations and tailor specific treatment strategies accordingly.
Marc K. Hellerstein, MD, PhD from the University of California, Berkeley. The project is entitled “Lifespan of SARS-CoV-2 reactive T-cells in COVID-19: Rationale for T-cell based vaccines”
Effective vaccines will be central to managing the world-wide COVID-19 crisis. Although both humoral and cell-mediated immunity play key roles in protective immunity against intracellular infections like viruses, current vaccine efforts for SARS-CoV-2 are generally focusing on antibody response and on the spike protein as immunizing antigen. Available data justifies emphasis on SARS-CoV-2 reactive T-cells to promote durable protective immunity and minimize pathogenic immunity.
Our goal is to characterize the durability of SARS-CoV-2 virus-reactive T-cells after natural COVID-19 infection and lay the groundwork for assessing T-cell immune responses to SARS-CoV-2 vaccines.
Aim 1. Will establish a clinical assay for monitoring in vivo lifespan of SARS-CoV-2 reactive CD8 T-cells in newly diagnosed patients and lay the groundwork for evaluating SARS-CoV-2 vaccines.
Aim 2. Will determine the durability of SARS-CoV-2 reactive CD8 T-cells after natural infection in COVID-19 patients.
William H. Hildebrand, MA, PhD from University of Oklahoma Health Sciences Center. The core is entitled “HLA Typing Core for CCHI Investigators.”
Our laboratory at OUHSC pioneered Human Leukocyte Antigen (HLA) typing by DNA sequencing. We provide CLIA-certified and ASHI-accredited high resolution typing of HLA for clinical transplantation and NIH grantees that study infectious disease and vaccine development. In 4¾ years, we have completed 10,537 HLA typings for CCHI investigators.
(Our typing lab has evolved from a research pedigree; NIH has allowed us to complete proteomics-based studies whereby we gather HLA proteins from pathogen-infected cells to determine how infection influences immunity. Today, our HLA typing services are positioned to strengthen human immunology research studies of infectious disease and vaccine development, and support hypothesis-driven stand-alone funding mechanisms.
Troy D. Randall, PhD from University of Alabama at Birmingham. Project is entitled “Lung-resident memory B and T cells from COVID19 convalescent patients.”
Given the respiratory tropism of SARS2, immunity should be targeted to the lung and airways. In this regard, many studies show that lung-resident memory T cells are an essential component of respiratory immunity. Moreover, some lung-resident T cells reside in the airways, where they act as first responders to secondary infections. We recently identified lung-resident memory B cells, and showed that they reside in the lung without recirculating, express a unique array of homing receptors and are first responders to secondary infections. Our new data also show that many lung-resident memory B cells are in the airways and it is these cells that first respond to secondary infection.
Unfortunately, we know very little of lung-resident memory B cells in mice and nothing at all about them in humans. Our hypothesis is that SARS2-specific lung-resident memory B and T cells will be phenotypically, functionally and clonally distinct from their counterparts in the circulation.
To test this hypothesis, we will acquire cells from the airways of SARS2-convalescent patients by bronchoalveolar lavage (BAL) and compare them to their circulating counterparts using single cell RNAseq coupled with BCRseq and TCRseq. We will also quantify SARS2-specific IgM, IgG and IgA in serum and BAL fluid to identify differences in specificity, cross-reactivity, and isotypes between locations. We believe our experiments will significantly advance human immunology because they will directly characterize antigen-specific, lung resident memory B and T cells responding to an important and deadly human pathogen.
Stephanie Boisson-Dupuis, PhD from Rockefeller University applied from the Rockefeller University CCHI U19 Center (Ravetch, PD/PI). Project is entitled “Inherited human PD-1 deficiency”
We identified by whole exome sequencing (WES), a patient with TB and a homozygous mutation in PDCD1, encoding PD-1. The patient suffered from extra-pulmonary TB as well as autoimmune diabetes and hypothyroidism. We hypothesize that inherited PD-1 deficiency underlies both the infectious and autoimmune clinical phenotypes of this patient. We aim to characterize this patient with inherited PD-1 deficiency by tackling three specifics aims:
(i) To determine the molecular genetic and biochemical basis of PD-1 deficiency in this family, in the context of the population genetics at the PDCD1 locus.
(ii) To determine the development of myeloid and lymphoid leukocyte subsets, including in particular T and B cell subsets that normally express PD-1.
(iii) To determine the function of these leukocyte subsets, in relation to infection and auto-immunity, in various experimental conditions.
Stylianos Bournazos, PhD from Rockefeller University applied from the Rockefeller University CCHI U19 Center (Ravetch, PD/PI). Project is entitled “Modulating the antibody response to vaccination through targeting the CD40 axis”
With the ultimate goal of identifying targets that could improve annual influenza vaccination, the overall objectives in this application are to determine the role for CD40 signaling in the post-translational modification of antigen-specific IgG in vitro and in vivo. We will pursue two specific aims:
1) Define the role for CD40 signaling in the control of antibody glycosylation in vitro.
2) Determine whether or not CD40 signaling can alter antigen-specific IgG structure in vivo in humans.
Shirit Einav, MD from Stanford University applied from the Stanford University CCHI U19 Center (Davis, PD/PI). Project is entitled “Deciphering the pathogenesis of severe dengue in natural infection in children via single-cell approaches”
We will use available samples from our Colombia cohort and two complementary single-cell strategies to monitor host immune responses to DENV infection and identify predictive biomarkers of severity. The Colombia cohort consists of 200 children, 80 adults and 60 healthy controls. Inclusion criteria: 2 years old and above, fever for 1-5 days, and positive for DENV antibody or antigen. Patients displaying SD upon presentation were excluded. We collected whole blood, serum, and PBMC samples at various time points during the disease course and upon convalescence. Symptoms, signs, and lab studies were documented by clinical experts. We confirm the diagnosis of dengue and distinguish primary from secondary infection.
Aim 1. Utilize and further the viscRNA-seq technology to profile gene expression at a single-cell level in distinct cell populations in natural dengue infection in children. A. We will profile transcriptomic responses in distinct cell populations in 12 gender-balanced samples collected upon presentation from patients who are 5-10 years old.
Aim 2. Profile the immune responses and functional states in PBMC samples from children with dengue via CyTOF. To correlate the transcriptomic data with functional immune phenotypes, we will characterize the immune state of cell subsets by analyzing protein expression via CvTOF using our panel of markers.
William Hildebrand, PhD, MA from the University of Oklahoma Health Sciences Center applied from the Oklahoma Medical Research Foundation CCHI U19 Center (Coggeshall, PD/PI). The Core is entitled “HLA Typing Core”
Our Laboratory at the University of Oklahoma Health Sciences Center (OUHSC) pioneered Human Leukocyte Antigen (HLA) typing by DNA sequencing and we continue to provide CLIA (Clinical Laboratory Improvement Amendments) certified and ASHI (American Society for Histocompatibility and lmmunogenetics) accredited high resolution typing of HLA for clinical transplantation and NIH grantees that study infectious disease and vaccine development.
Our U19 service core offers typing of traditional class I and II MHC, KIR, and non-classical MHC molecules. In the past four years the sample processing requested most frequently by CCHI investigators has been high resolution typing at the HLA-A, -B, -C, -DRB1, -DPB1, – DQB1, and -DQA1 gene loci. Because U19 investigators occasionally request non-classical MHC and KIR typing, we will continue to provide this service as well as clinical grade typing for clinical studies that may arise.
Kate Jeffrey, PhD from Massachusetts General Hospital applied from the Massachusetts General Hospital/University of Pennsylvania CCHI U19 Center (Chung and Wherry, PD/PIs). Project is entitled “Examination of the Epigenome in Single and Low Number Human Immune Cells”
AIM 1: ASSESS CHROMATIN ACCESSBILITY AND HISTONE MODIFICATIONS IN HBV VIRUS-SPECIFIC CD4 VERSUS CDS RESPONSES.
We will use leukapheresis samples from chronic HCV patients pre and post antiviral therapy to establish single cell and small bulk population epigenetic studies, before we will apply the new technology to our current study analyzing the impact of PD-1 blockade on HBV-specific T cells in blood and liver of chronic HBV patients.
AIM 2: DEFINE THE MACROPHAGE EPIGENOME AND FUNCTION IN CHRONIC VIRAL INFECTION.
(i) Using Fine Needle Aspiration Biopsy (FNA) and PBMCs from patients with chronic HCV entering a novel clinical trial of checkpoint inhibitor therapy, we will assess chromatin accessibility and histone modifications in bulk and single cell and whether PD-1 treatment shifts the macrophage epigenetic landscape toward a pro-inflammatory and anti-viral phenotype. (ii) Beginning with CD14-sorted PBMCs from patients with chronic hepatitis C who were participants in our HCV treatment trial, we will specifically apply bulk and single cell ATAC-Seq, Mint-ChlP and CUT&TAG to stored leukapheresed PBMC samples (50-100,000 cells) from 22 patients collected prior to and 12 weeks following completion of curative antiviral therapy for HCV. Once conditions for single cell and small bulk populations are refined, then the technique will be applied to frozen, stored macrophage populations from liver FNAs performed in the same patients. We will CD14 sort these aspirates, perform bulk and single cell ATAC-Seq and CUT&TAG (50-10,000 cells) and compare outputs from FNA to PBMC compartments.
Murali Krishna Kaja, PhD from Emory University applied from the Emory University CCHI U19 Center (Ahmed, PD/PI). Project is entitled “Characterizing dengue specific IgG subclass antibody responses and Fc glycosylation changes during primary and secondary dengue infections in India”
We propose to analyze if there is any correlation between the disease severity and lgG subclass of the dengue specific antibody or their Fc glycosylation status in patient samples depending upon their primary versus secondary dengue infection status. The following specific aims are proposed:
Aim 1. Compare relative abundance of dengue specific plasma lgG subclasses in pediatric cohort from lndia with and without severe disease in primary and secondary dengue infections.
Aim 2. Compare lgG Fc glycosylation changes in pediatric cohort from lndia with and without severe disease in primary and secondary dengue infections.
We will address these questions by characterizing dengue specific lgG subclasses in plasma and their Fc glycosylation status and correlating these changes with the status of primary versus secondary dengue infections, disease severity, neutralizing antibody responses and infecting virus serotype.
Rodney King, PhD from the University of Alabama at Birmingham applied from the University of Alabama at Birmingham CCHI U19 Center (Lund, PD/PI). Project is entitled “The influence of isotype CH1 on antigen binding”
The first constant domain of IgG1-4 is highly conserved, however, the CH1 sequence of IgM, IgA, and IgD differ significantly, both from one another as well as those of IgG. Despite their high degree of conservation, IGHC1 region of IgG1 and IgG3 can specifically influence the reactivity of otherwise identical recombinant Abs. The potential influences of IGHC1 domains of non-IgG isotypes on Ab fine specificity and affinity have not been systematically examined. We hypothesize that differences in IGHC1: 1) affect antigen reactivity by influencing variable region structure; 2) are more pronounced between IgM, A, and D than IgG1-4; 3) are more frequent within the glycan reactive repertoire; and 4) serve to restrict the isotype distribution of certain B cell clonotypes.
We will directly assess the role of IGCH1 on antigen binding. We will generate a series of recombinant human antibodies, reactive with a T-dependent and T-independent antigen, as hybrid IGHC1 antibodies (replace the IGHC1 of IgG1 with IGHC1 of IgM, IgD, IgA, and IgG2) to compare the antigen reactivity of these human Abs differing only in their CH1 to determine any influence of isotype specific IGHC1 in Ab binding.
Holden Maecker, PhD from Stanford University applied from the Stanford University CCHI U19 Center (Davis, PD/PI). Course is entitled “CyTOF Immune Monitoring Course”
The most requested technology training in the last several years has consistently been for CyTOF. We propose to hold a 2.5-day “CyTOF and Immune Monitoring” Course at Stanford in the Spring of 2020 and 2021, with up to 24 enrollees for the lab component (additional enrollees can be accepted for the lectures). The course will focus heavily on CyTOF mass cytometry, with three mornings of lectures and two afternoons of hands-on lab work in this area. This will be well-supported by the Maecker lab’s two instruments and 1200 square foot lab in the Fairchild Science Building on the Stanford campus.
The course will build on the successful format of previous Nolan-Maecker courses, with morning lectures and afternoon hands-on lab sessions. The lab sessions will focus exclusively on CyTOF (sample preparation, acquisition, and analysis). The lectures in the first two days will also be CyTOF-centric, covering practical aspects like sample barcoding, quality control, analysis methods, etc. The last morning, lectures on other technologies (e.g., Luminex/Olink, TCRseq, MIBI/CODEX, ATACseq) will be given.
Anoma Nellore, MD from the University of Alabama at Birmingham applied from the University of Alabama at Birmingham CCHI U19 Center (Lund, PD/PI). Project is entitled “Characterization of Mucosal and Circulating HA-specific B cells after LAIV”
We propose to identify HA-specific B cell subsets responding to LAIV in tonsils, determine their relationship to HA-specific B cell subsets in the blood amd define which of these cell types contribute to recall responses to vaccination in the following year.
AIM 1. To identify the relationships between HA-specific B cells in tonsil and blood after LAIV vaccination.
AIM 2. To identify which HA-specific B cells from tonsil and blood are recalled as ASCs after Ag rechallenge.
Our studies will define how B cells stimulated by vaccination in lymphoid tissues are related to B cells in peripheral blood at the time of immunization and a year later in recall responses. As a result, our data will define a cellular signature of vaccine efficacy and longevity in peripheral blood and may even identify the first correlate of protection for LAIV. Because most vaccine studies in humans are performed using samples of peripheral blood, our data will also advance the development of other vaccines by giving investigators a cellular signature in peripheral blood that gives us insight into what is happening in lymphoid tissues.
Catherine Wu, MD from the Dana-Farber Cancer Institute applied from the Massachusetts General Hospital/University of Pennsylvania CCHI U19 Center (Chung and Wherry, PD/PIs). Project is entitled “Tissue localization of virus-specific T cells by spatial sequencing technologies”
Slide-seq is a new method that spatially resolves transcriptome sequencing data. We propose applying TCR amplification to cDNA libraries generated by the Slide-seq protocol. Further, we will adapt Slide-seq to sensitively detect targeted transcripts. Thus, we propose to develop a TCR Slide-seq technology that spatially localizes antigen-specific T cells within specimens via their TCR sequences (Aim 1), and determines the T cells’ functional and differentiation states, specificity for common viruses, and proximity to other immune cell types (Aim 2).
Aim 1. Develop Slide-seq to spatially locate T cells within biopsy specimens based on TCR sequence by combining rhTCRseq and Slide-seq technologies.
Aim 2. Measure expression of common viral genes and genes that are involved in T cell function and immune cell interactions.