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Updated: Apr 23, 2026
Visualizing Lymph Node Structure and Cellular Localization using Ex-Vivo Confocal Microscopy
Published on: August 9, 2019
The normal human lymph node cell classification and landscape defined by high-dimensional spatial proteomics
Maddalena M Bolognesi1,2, Lorenzo Dall'Olio3, Giulio Eugenio Mandelli4,5
1Istituto di Bioimmagini e Sistemi Biologici Complessi (IBSBC) - CNR Via F.lli Cervi, Segrate, Italy.
Insights
This study comprehensively maps all cells within human lymph nodes (LN) using advanced techniques, revealing 77 distinct cell types and novel spatial organization critical for immune responses.
Area of Science:
- Immunology
- Cell Biology
- Proteomics
Background:
- Lymph nodes (LN) are crucial secondary lymphoid organs (SLO) for immune responses.
- Traditional methods often disrupt LN spatial organization, limiting in situ analysis.
- A comprehensive spatial atlas of normal human LN cell types is needed.
Purpose of the Study:
- To spatially map and classify all cell types within normal human lymph nodes in situ.
- To identify novel cell subsets, interactions, and spatial niches within the LN microenvironment.
- To provide a high-dimensional proteomic dataset for understanding LN organization.
Main Methods:
- Utilized a 78-marker antibody panel with hyperplexed cyclic staining (MILAN) for in situ cell classification.
- Employed an analytical bioinformatics pipeline (BRAQUE) for data analysis.
- Analyzed 19 human lymph nodes free of pathology.
Main Results:
- Classified 77 distinct cell types, including T cells, B cells, innate immune cells, and stromal cells.
- Identified 27 unique T-cell subsets and novel B-cell types based on TCF7 and CD5 expression.
- Revealed previously unrecognized spatial distribution of mature B cells and type 2 conventional dendritic cells.
- Discovered novel cell-cell interactions and LN landscape niches through neighborhood analysis.
Conclusions:
- This high-dimensional proteomic interrogation provides an unprecedented spatial map of the normal human lymph node.
- The findings reveal novel cell types, interactions, and spatial organization critical for immune function.
- This resource facilitates deeper understanding of LN immunity and potential therapeutic targets.
Abstract:
Lymph nodes (LN) are key secondary lymphoid organs (SLO) for a coordinated immune response. They have been extensively characterized by numerous investigative techniques chiefly as single cell suspensions because they are composed of vagile yet crowded hematolymphoid elements, unfriendly to spatial tissue organization-saving techniques. We comprehensively classify in situ all cells of 19 human LN free of pathology with a 78-marker antibody panel, an hyperplexed cyclic staining method, MILAN, and an analytical bioinformatic pipeline, BRAQUE. A total of 77 cell types were classified, encompassing T, B, innate immune and stromal cells. CD4 and CD8 T-cells were classified into 27 unique subsets by leveraging the expression profiles of TCF7, the presence of co-inhibitory receptors and the spatial distribution. CD5 and TCF7 expression defined novel B-cell types. CD27 + mature B-cells occupied previously unrecognized nodal spaces non-overlapping with the cortex and the plasma-cell rich medullary cords. Type 2 conventional dendritic cells were located in nodular paracortical aggregates. Statistically controlled pairwise neighborhood analysis showed sparse cell-cell interactions, known and new neighbors, established and novel LN landscape niches. A high-dimensional proteomic interrogation of the normal human LN provides spatial allocation of known cell types, novel interactions and the landscape organization.
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