2021-2022

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.

2019-2020

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.