We propose a U19 Cooperative Center on Human Immunology at The Jackson Laboratory (JAX CCHI) to elucidate the innate immune networks that shape adaptive immune responses to respiratory viral infections in the human lung. Epithelial barriers lie at the interface between host and environment, where they sense invading pathogen. Dendritic cells (DCs) present pathogen-derived antigens to T and B cells to induce immune responses. However, the impact of the human lung tissue environment on DC and other cells, such as the newly identified innate lymphoid cell (ILC) family, as well as bacteria-reactive MAIT cells, is not completely understood. An understudied environmental factor is the lung microbiome. Microbiota are known to critically modulate the function of immune cells, particularly at mucosal surfaces, but how this occurs in the lung is not fully addressed. The JAX CCHI seeks to address these critical questions using a multi-disciplinary experimental approach that will integrate immunology with epithelial cell biology along with genomic, cellular, functional and microbiome parameters identified in human lung tissues. Our overarching hypothesis is that the quality and magnitude of mucosal T cell responses to respiratory viral infections are determined by the cross- talk between microbiota, epithelial cells and leukocytes. To address this hypothesis, we structured the JAX CCHI around two integrated research projects focused on basic immunological mechanisms of lung antiviral immunity; a technology development project that will create sophisticated cellular models leveraging 3D bioprinting, gene editing tools and microbiome-immune assays to support project objectives; a sample core for storage and distribution of human tissues; and a microbiome core for specialized microbiome profiling, cultivation, and computational analysis. Our Center will bring together clinicians with experts in lung immunology, the microbiome, bioengineering, genomics and computational biology to achieve our goals and maximize the potential of this research. An administrative core will provide coordination, communication and oversight for the program. The goals of this CCHI are to: 1) Understand how the networks of epithelial cells and immune cells in the human lung regulate innate and adaptive immunity to respiratory viruses; 2) Define how inflammation driven by the microbiome dictates the steady state of tissue, i.e., immune set-point; 3) Determine if and how this immune set-point is altered in two pulmonary diseases, childhood asthma and adult lung cancer, which have a major impact on public health; and 4) Develop innovative technologies to model human lung-immune dynamics and elucidate molecular mechanisms, cell types and pathways key to lung antiviral responses. Impact: Through studies focused on the sensors, inducers and modulators of antiviral immunity in the human lung, our CCHI will contribute insights that could help improve outcomes for infectious and other immune diseases that originate in or secondarily impact the lung.
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Our U19 Center consists of three Cores and three Projects:
ADMINISTRATIVE CORE
PALUCKA, ANNA KAROLINA, Core Lead
The goal of the Administrative (Admin) Core is to provide management, oversight and logistical support for the proposed JAX Collaborative Center on Human Immunology (JAX CCHI). The essential functions of the Admin Core are to: 1) facilitate communication and foster a strong collaborative environment among the PIs/Investigators; 2) establish the External Scientific Advisory Group (ESAG), coordinate annual ESAG meetings and Implement their recommendations for improvement or advancement; 3) maintain alignment of the JAX CCHI with the goals and objectives of the broader CCHI program; 4) monitor the progress of the research and technology development projects and cores towards their specific research and resource development goals; 5) optimize resource allocation and fiscal management; 6) ensure swift resolution of any logistic, scientific and fiscal challenges; and 7) ensure efficient data sharing through ImmPort. JAX is home to several NIH-funded Research Centers and has a long history of successful contributions to NIH- funded consortium efforts. JAX also offers excellent capabilities, facilities and resources to support the Admin Core and the JAX CCHI as a whole. The Admin Core will be led by Center Director Dr. Karolina Palucka, an accomplished human immunologist with significant experience in center leadership, and supported by an experienced and dedicated Program Manager. The Admin Core will promote the goals of the JAX CCHI and the broader program effort through the following Specific Aims: Aim 1. Optimize integration and function of JAX CCHI activities through strong administrative and scientific oversight. Aim 2. Enhance communication and collaboration among JAX CCHI investigators. Aim 3: Ensure secure data management and efficient data dissemination via ImmPort. Aim 4. Manage resource allocation and fiscal accountability.
MICROBIOME CORE
OH, JULIA S, Core Lead
The microbiome is a critical factor in educating both systemic as well as local immunity. Compartmentalized immune interactions with the resident flora have been identified at barrier tissues of the body, including the skin, gut, and lung. Each of these niches has different microbes and microbial community characteristics, which in turn uniquely shape and maintain innate and adaptive responses. In the lung, host immune responses to pathogen colonization—whether bacterial, fungal, or viral—are highly influenced by the local microbiota. A central hypothesis of this center is that the activity and specificity of the lung immune response can be amplified or diminished based on microbial configurations. Thus, to understand the milieu and activity of tissue-resident lung cells, including macrophages, lung dendritic cells, and lymphocytes, we must investigate the composition and functional interactions of the local microbiome with the host tissue. The central goal of the Microbiome Core is to enable discovery of mechanisms by which the microbiome influences lung immune function, as per the goals of The Jackson Laboratory Cooperative Center on Human Immunology (JAX CCHI). The Microbiome Core will be closely integrated with the Sample Core, providing sequence data, microbial cultivars, and computational analyses for both Research Projects and the Technology Development project. The Microbiome Core provides unique experimental and computational infrastructures that are not available as commodity services within or outside of The Jackson Laboratory. Key innovations that the Core brings to the JAX CCHI include: state-of-the-art metagenomic shotgun sequencing technologies and analyses at species- and strain-resolution; functional reconstructions; associative analyses with immune phenotypes; low-cost, microfluidics-based microbial isolate genome extraction; and MALDI (Matrix Assisted Laser Desorption/Ionization) technology for rapid assessment of strain identity and diversity. The application of these technologies to the goals of the JAX CCHI will be organized around three Specific Aims: AIM 1. Reconstruct microbial community composition from clinical samples at high resolution. AIM 2. Systematic cultivation of microbiota. AIM 3. Computational prediction of microbiome-immune interactions. Impact: The Core’s experimental and computational infrastructure will provide both the associative and mechanistic underpinnings for the CCHI’s central goals of understanding how the microbiome may modulate the lung’s immune response to viral perturbation.
SAMPLE PROCUREMENT AND MANAGEMENT CORE
PALUCKA, ANNA KAROLINA, Core Lead
The main function of Sample Procurement and Management Core (SPMC) is to provide Research Projects 1 and 2, the Technology Development project and the Microbiome Core with human specimens to support the proposed research of this CCHI. The SPMC has four Specific Aims: AIM 1: Finalize program-specific operations to receive and manage samples. AIM 2: Oversee sample collection, acquisition and processing. AIM 3: Manage sample storage, tracking and distribution. AIM 4: Oversee personnel training and compliance.
MODULATION OF VIRAL ANTIGEN PRESENTATION IN THE LUNG
PALUCKA, ANNA KAROLINA, Project Lead
Project 1 will elucidate how the networks of antigen presenting cells (APCs) in the human lung regulate immunity to respiratory viruses. The goal is to also explain how microbiome-driven lung inflammation or inflammation that is linked with neoplastic processes affects such responses. Project 1 is founded on the scientific premise that identifying the pathways underpinning immune cell responses to respiratory viruses in situ is key to identifying targets and strategies for designing and developing improved vaccines. Our overall hypothesis is that the lung environment defines immunological status, i.e., the “immune set-point”, and the function of tissue-resident dendritic cells (DCs). We further posit that the immune set-point impacts the fate of antigen and the quality and magnitude of ensuing mucosal T-cell immunity. Implicit in this hypothesis is a role for the local microbiome, which we predict contributes to the immediate environment by direct and indirect crosstalk with immune cells and ensuing inflammatory responses. We propose three aims: Aim 1 will test the hypothesis that steady state cellular and molecular networks in human lung tissue regulate the early response to respiratory viruses. We will define the composition and functional status of human lung tissue across a range of clinical situations: normal lung, uninvolved cancer patient lung and cancer-involved lung tissue. Correlative analyses with upstream environmental regulators such as the microbiome will identify pathways that control the magnitude and quality of ensuing adaptive immunity. Aim 2 will test the hypothesis that the generation of anti-viral T-cell immunity is modulated by lung epithelial cell (EC)-DC crosstalk and that this crosstalk is further modulated by commensal bacteria. We will determine how lung DCs exposed to virally-infected lung alveolar epithelial cells (AECs) modulate the differentiation of T cells; we will establish the molecular programs in DCs triggered by lung ECs that can explain T-cell phenotypes; and we will determine how the bacteria cultured from the mouth and upper respiratory tract impacts lung ECs and downstream responses. Aim 3 will test the hypothesis that the lung microenvironment modulates the cross-presentation capacity of lung-resident APCs thereby dictating the fate of viral antigen-specific CD8+ T cells. We will assess viral distribution and cross-presentation in the context of resistant and susceptible cells defined by expression of a viral resistance gene, Rab15; and the access of opsonized virus to cross-presenting compartments in lung myeloid cells. Thus, this project will elucidate the key innate immune networks that determine the overall outcome of adaptive immune responses during respiratory viral infections. Along with other Projects, our proposed research has a high potential to discover novel target molecules that will eventually help us design improved therapeutics and vaccines for respiratory infections. Finally, the work proposed here will guide development and validation of 3D printed lung tissues, which will enable genetic experiments and possibly future studies on human tissue immunity.
IMPACT OF AIRWAY EPITHELIUM ON INNATE AND ADAPTIVE IMMUNITY IN THE LUNG
COLONNA, MARCO, Project Lead
Respiratory viruses, such as respiratory syncytial virus (RSV), are major triggers of asthma exacerbation in children and adults. The overarching hypothesis of this project is that airway epithelial cells (AECs) coordinate responses to respiratory virus infection and aeroallergens. Moreover, intrinsic differences between AECs of asthmatic and healthy children change both innate and adaptive responses during infection and exposure to allergens. We hypothesize that infection of AECs from asthmatic children with RSV or HRV, in combination with allergen exposure, induces resident innate cells, including dendritic cells (DCs) and innate lymphoid cells (ILCs), to trigger type-2 responses. We will test this idea using AECs isolated from asthmatic and healthy children, grown at air/liquid interface (ALI) and infected with respiratory viruses in the context of allergen challenge. We will examine the response of DCs and ILCs co-cultured with these AECs, and their role in promoting type-2 inflammation through the following aims: Aim 1. Test hypothesis that AECs control local DC maturation and function. Since AECs in asthmatics may be “leaky” due to barrier defects, we will first determine whether more antigen is transferred by asthmatic than healthy AECs to DCs using modified RSV strains. Then, we will test whether AECs from asthmatic children differ in promoting DC differentiation and function, and how virus infection and allergen exposure impact this interaction. Finally, we will assess the ability of DCs exposed to AECs from asthmatic children to drive CD4 T cell differentiation and proliferation in co-culture assays. Aim 2. Test hypothesis that after allergen exposure and viral infections, asthmatic and healthy AECs differentially impact lung ILC2 through lipid mediators and vasoactive intestinal peptide. We will assess the role of AECs in regulating ILC2s, which are the most abundant ILCs in the lung. We expect to find that AECs from asthmatic children enhance ILC2 function during viral infection and allergen challenge. In our preliminary data, we found two distinct subsets of human lung ILC2s that differentially express receptors for oxysterols, retinoic acid, and vasoactive intestinal peptide. Thus, we propose to define the impact of these lipidic and neuropeptide mediators on the activation of different ILC2 populations. Aim 3. Test hypothesis that ICOS+ ILCs regulate lung immune responses. We made the novel observation that the lung contains an unusual subset of ICOS+ ILCs. These cells do not fit into the ILC1, ILC2, ILC3 paradigm, express a marker of regulatory lymphocytes and are enriched in an immunosuppressive environment associated. Thus, we hypothesize that these cells may have regulatory functions. To test this, we propose to isolate lung ICOS+ ILCs and define their transcriptome profile ex vivo as well as their functional capabilities in vitro, including cytokine and chemokine secretion. Moreover, we propose to test their responsiveness to stimuli derived from ALI cultures exposed to HDM and/or viruses.
DEVELOPMENT OF 3D ALVEOLAR TISSUE MODELS, CRISPR-EDITING AND MICROBIOTA-IMMUNE RESPONSE ASSAY PLATFORMS FOR DECIPHERING HUMAN LUNG IMMUNITY
UNUTMAZ, DERYA, Project Lead
The goal of the Technology Development project (Tech Dev) is to develop approaches, tools and assays that address the needs of The Jackson Laboratory Cooperative Center on Human Immunology (JAX CCHI) and that advance the capabilities of the scientific community to tackle questions regarding human lung immunity, human immune-microbiota interactions and basic mechanisms of immune cells. Major questions related to lung immune function remain unanswered—such as the cell-to-cell interactions between immune and lung epithelial cells that shape responses to foreign agents, or how the presence of microbiota in the airways or within lung compartments influences the pathogenesis of viral infections and other lung diseases. A significant technical barrier to studying human immune-lung dynamics is the sheer complexity of the human lung—which constantly filters airborne particles, infectious organisms and air through dynamic interactions between the lung epithelium and resident immune cells such as macrophages or dendritic cells. This complexity cannot be easily modeled in animal systems or using deceased human lung tissue. To surmount these challenges, Tech Dev will focus on three innovative human tissue platforms: 1) three-dimensional (3D) bioprinted models of the lung and upper respiratory environment for investigating the functional lung-immune interactome during exposure to viral or metabolic agents; 2) CRISPR/Cas9-based tools to genetically engineer primary human immune cell subsets, hematopoietic stem cells and/or lung epithelial progenitors to probe cell function; and 3) a functional in vitro platform for screening lung-resident microbiota and determining their impact on human lung immune responses. Each of these platforms addresses a specific unmet need in the application and will enable us, respectively, to study the human lung immunity within a dynamic and physiologically relevant microenvironment, to interrogate specific cell types and molecular pathways predicted to respond to viral infections, and to assess the impact of bacterial metabolites isolated from human airways on antiviral responses. Through these efforts, the JAX CCHI will be equipped to address previously inaccessible questions related to lung-immune dynamics, towards a more mechanistic understanding of lung immune function. Our Specific Aims are: Aim 1. Develop in vitro models of human lung tissue-immune interactions using 3D bioprinting. Aim 2: Optimize CRISPR-based genetic tools for use in engineering primary human immune and lung epithelial cells. Aim 3: Develop a functional immune assay platform to determine the immunomodulatory landscape of human lung and airway microbiota.
