The normal human lymph node cell classification and landscape defined by high-dimensional spatial proteomics
Open accessPublished version of a preprint
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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- Authors
- Bolognesi, M. M., Dall’Olio, L., Mandelli, G. E., Lorenzi, L., Bosisio, F. M., Haberman, A. M., Bhagat, G., Borghesi, S., Faretta, M., Castellani, G., & Cattoretti, G.
- Affiliations
- National Research Council (IT)
- Istituto delle Scienze Neurologiche di Bologna (IT)
- Azienda Socio Sanitaria Territoriale degli Spedali Civili di Brescia (IT)
- Universitair Ziekenhuis Leuven (BE)
- Yale University (US)
- NewYork–Presbyterian Hospital (US)
- University of Milano-Bicocca (IT)
- European Institute of Oncology (IT)
- University of Bologna (IT)
- Corresponding author
- Cattoretti, Giorgio
- ORCID
- Venue
- PLOS One
- Publisher
- Public Library of Science (PLoS)
- Published
- 2026-04-21
- Volume
- 21
- Issue
- 4
- Pages
- e0346693
- ISSN
- 1932-6203 (ISSN-L)
- OpenAlex
- W7155090949
- Cited by
- 2
- References
- 7
- Citations by year
- 2026: 2
- FWCI
- 4.09
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- PLoS ONE (publishedVersion, cc-by)
- bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- BOA (University of Milano-Bicocca) (submittedVersion, cc-by)
- Lirias (submittedVersion, cc-by)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
- Institutional Research Information System (Università degli Studi di Brescia) (submittedVersion)
- Funders
- Regione Lombardia (POR FESR 2014–2020, Call HUB Ricerca ed Innovazione: ImmunHUB)
- Horizon 2020 Framework Programme (EU Horizon 2020 programme GenoMed4All project #101017549, HARMONY and HARMONY-PLUS project #116026)
- Fondazione AIRC per la ricerca sul cancro ETS (projects #26216)
- Universitaire Ziekenhuizen Leuven, KU Leuven (INTERNE FONDSEN MIDDEL-Zware infrastructuren EMH-D8191-AKUL/19/30 I005920N, FWO Fundamenteel Klinisch Mandaat EMH-D8972-FKM/20)
- European Research Council (Project code CN_00000033, Concession Decree No. 1034 of 17 June 2022)
- Consiglio Nazionale delle Ricerche (“National Biodiversity Future Center - NBFC”.)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Single-cell and spatial transcriptomics
- Life Sciences › Immunology and Microbiology › Immunology › T-cell and B-cell Immunology
- Physical Sciences › Chemistry › Spectroscopy › Advanced Proteomics Techniques and Applications
- Keywords
- human lymph nodes, spatial proteomics, secondary lymphoid organs, CD4+ T cell subsets, CD8+ T cell subsets, TCF1, B cell subsets, CD5+ B cells, type 2 conventional dendritic cells, cell-cell interactions, lymph node architecture, stromal cells
- MeSH terms
- B-Lymphocytes, Dendritic Cells, Humans, Lymph Nodes, Proteomics
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