# Project 3 - Lymph Nodes

> **NIH NIH U54** · UNIVERSITY OF FLORIDA · 2020 · $547,554

## Abstract

The lymph node serves as an in-line filter of the lymphatic and interstitial circulation, containing fluids, particulates
and cells, in particular dendritic cells (DCs), while also receiving leukocytes from the circulatory system, ultimately
for the purpose of systemic immune surveillance against infectious agents and malignancy. Given these key
physiological functions, we deem it important to develop a three-dimensional (3D) biomolecular tissue map of
the normal human lymph node (Organ Specific Project 3 [OSP 3]). With over a decade of experience in the
procurement of transplant-quality organs for research, including those of the lymphatic system, approximately
600 human lymph nodes banked in our biorepository, and a robust research pipeline, we are well equipped to
evaluate in 3D space, specific cellular interactions, gene and protein expression patterns, as well as protein
modifications that govern regional immune activation, anergy, and tolerance activities in this secondary lymphoid
organ. To achieve this goal, we intend to perform systematic, anatomic dissection of freshly acquired human
lymph nodes, with assessment of normality by an independent pathologist, to support all subsequent AIMs (AIM
I). We will generate macro images of the intact organ and lymph node sections (AIM IIA) to guide microanatomic
analyses. Our microscopic imaging platforms (AIM IIB-D) include immunohistochemistry (IHC) and
immunofluorescence (IF) with widefield, confocal, multiphoton, and Stochastic Optical Reconstruction
Microscopy (STORM) imaging, along with optical clearance with light sheet fluorescence microscopy (LSFM)
and Pathology-optimized Expansion Microscopy (ExPATH). These applications will then be coupled with
histology topography cytometry analysis toolbox (histoCAT), FluoRender and Amira-Avizo Software to enable
3D reconstruction (Data Core). Dispersed lymph node cells will be immunophenotyped and isolated by
fluorescence activated cell sorting (FACS) to characterize the normal T cell receptor (TCR) and B cell receptor
(BCR) repertoire (AIM III). Biomolecular analysis of tissue sections by imaging mass cytometry (IMC) (AIM IVA)
offers the ability to simultaneously visualize dozens of proteins, their modifications, and RNA species at the
cellular level with 3D reconstruction. This, coupled with single molecule fluorescence in situ hybridization
(smFISH) analyses and single cell RNA-Seq using 10x GENOMICS GemCode and 10x Chromium Technology
(AIM IVB) could facilitate discovery of novel cellular markers relevant to human lymphatic anatomy and
immunological function. These novel markers will then after validation be applied to the optical and IMC platforms
for 3D location within the lymph node. Both raw and analyzed data generated at in each stage of the research
pipeline will be integrated into the HIVE via interactions with the Data Core, the Coordination Core, and other
TMC efforts. We believe these proposed studies represent a critical step toward a hi...

## Key facts

- **NIH application ID:** 9970202
- **Project number:** 5U54AI142766-03
- **Recipient organization:** UNIVERSITY OF FLORIDA
- **Principal Investigator:** Clive H Wasserfall
- **Activity code:** U54 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $547,554
- **Award type:** 5
- **Project period:** 2018-09-14 → 2022-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9970202

## Citation

> US National Institutes of Health, RePORTER application 9970202, Project 3 - Lymph Nodes (5U54AI142766-03). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9970202. Licensed CC0.

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