The lymphatic system serves five primary roles: 1) removal of excess body fluids; 2) absorption and transport of fatty acids/chyle to the circulatory system; 3) blood filtration; 4) mounting the primary defense against infections and cancer through immune cell production and activation; and 5) generation and activation of regulatory immune cells that protect against autoimmune/autoinflammatory disease. Given these key physiological functions, we deem it important to develop a three-dimensional (3D) tissue map for three major lymphoid organs of the human immune system: spleen, thymus and lymph nodes (Organ Specific Projects 1- 3, respectively). As one of our primary strengths, we have over 10 years of experience in the procurement of transplant-quality organs for research, including those of the lymphatic system. As part of this 24/7/365 effort, we have well-established relationships with the U.S. Organ Procurement Organizations (OPO), having referrals from 56/58 over the last decade. Our standard operating procedures (SOPs), both published and web-based, include assessment of normality, quality control assays, and systematic anatomical dissection/storage. We also participate in National QA/QC programs to evaluate tissue processing/banking procedures. Our approach to developing assay pipelines towards the common goal of a 3D tissue map will initially involve acquiring a macro image of the intact tissue and addressing tissue morphology, using our strengths in magnetic resonance imaging (MRI). The proposed optical microscopy pipeline will address microanatomical features using formalin fixed paraffin embedded (FFPE) and optimal cutting temperature (OCT) compound embedded sections as well as tissue optical clearing and expansion. These specimens will be studied from nm to mm resolution using stochastic optical reconstruction microscopy (STORM), confocal, multiphoton and light sheet fluorescence microscopies (LSFM), with all pipelines sharing a common file format for simplified 3D reconstruction. Based on the unique role for lymphatic organs in production and trafficking of immune cells, fluorescence activated cell sorting (FACS) of cells from blood and each lymphatic organ will provide a comparison of the patient- specific immune cell repertoire and serve in subsequent single cell RNA-seq analyses. To co-register biomolecules to their cognate cells, we will employ imaging mass cytometry (IMC), multiplex single molecule fluorescence in situ hybridization (smFISH) and multiplexed error-robust FISH (MERFISH) to map cellular protein and mRNA expression, ultimately on each 3D tissue atlas. Our experience in organ procurement, the availability of the National High Magnetic Field Laboratory (NHML) Advanced Magnetic Resonance Imaging and Spectroscopy (AMRIS) Facility at UF, multiphoton/confocal microscopy, LSFM, state of the art FACS, 10X GENOMICS Chromium controller, and high throughput cDNA library sequencing for RNA-Seq at the University of Florida, as well as world leading IMC and 3D data analysis facility at the University of Zurich, ideally positions us to provide the highest quality 3D Human BioMolecular Atlas (HuBMAP) of the lymphatic organs.
With similarities to the circulation, the organization and function of the lymphatic system stretches across the entire human body. It is critical in controlling body fluids, blood filtration, mounting a defense against infections and cancer, as well as regulating immune cells to protect against autoimmune/autoinflammatory diseases. Tissue organization and function are directly linked in spleen (blood filtration), thymus (T lymphocyte development) and lymph nodes (immune cell priming and activation for local/peripheral immunity) Developing three-dimensional (3D) tissue maps of these normal immune organs, across decades of the human life span, would provide novel insights that would improve our understanding of the immune system structure and function including the trafficking of white blood cells and the immune surveillance mechanisms toward combating infectious insults. We plan to obtain a macro image of each tissue with high-resolution magnetic resonance imaging (MRI) to serve as the guide on which our optical microscopy pipeline will provide a detailed microanatomical description of each tissue, from nanometer to millimeter resolution. We will then co-register the expression of proteins and mRNAs onto our 3D tissue maps with state of the art imaging mass cytometry and multiplexed, high throughput in situ hybridization. All of our optical imaging modalities and biomolecule co- registration studies will utilize a common file format for 3D reconstruction which will facilitate streamlined data sharing with the HuBMAP Integration, Visualization, and Engagement (HIVE), other HuBMAP Tissue Mapping Centers (TMCs), and the broader research community.
