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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\u2009+\u2009mature 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.<\/jats:p>","DOI":"10.1371\/journal.pone.0346693","type":"journal-article","created":{"date-parts":[[2026,4,21]],"date-time":"2026-04-21T17:53:23Z","timestamp":1776794003000},"page":"e0346693","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":1,"title":["The normal human lymph node cell classification and landscape defined by high-dimensional spatial proteomics"],"prefix":"10.1371","volume":"21","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-8238-2369","authenticated-orcid":true,"given":"Maddalena M.","family":"Bolognesi","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lorenzo","family":"Dall\u2019Olio","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Giulio Eugenio","family":"Mandelli","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Luisa","family":"Lorenzi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Francesca M.","family":"Bosisio","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ann M.","family":"Haberman","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6250-048X","authenticated-orcid":true,"given":"Govind","family":"Bhagat","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Simone","family":"Borghesi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mario","family":"Faretta","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gastone","family":"Castellani","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3799-3221","authenticated-orcid":true,"given":"Giorgio","family":"Cattoretti","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2026,4,21]]},"reference":[{"issue":"5","key":"pone.0346693.ref001","doi-asserted-by":"crossref","first-page":"409","DOI":"10.1080\/01926230600867727","article-title":"Normal structure, function, and histology of lymph nodes","volume":"34","author":"CL Willard-Mack","year":"2006","journal-title":"Toxicol Pathol"},{"issue":"5","key":"pone.0346693.ref002","doi-asserted-by":"crossref","DOI":"10.1242\/jcs.241828","article-title":"The lymph node at a glance - 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