Follicle-like niches outside the cortex? 3D phase-contrast µCT revealed medullary B cell nodules in mucosa-draining lymph nodes

Paul Schütz¹Florian Lennart Schwarzenberg^1*Lennert Jochen Weber¹Jörg Hammel²Bente Siebels³Paula Nissen³Bernd Walkenfort⁴Sarah C. Irvine²Jasmin Bartl¹Julia Herzen^5,6Christian Lohr⁷Clemens Wülfing^1*Stephan Henne¹

• ¹INI-Research, Group for Interdisciplinary Neurobiology and Immunology, University of Hamburg, Hamburg, Germany
• ²Institute for Materials Physics, Helmholtz-Zentrum Hereon, Geesthacht, Germany
• ³Section Mass Spectrometry and Proteomics, Center for Diagnostics, University Medical-Center Hamburg-Eppendorf, Hamburg, Germany
• ⁴Imaging Center Essen (IMCES), Electron Microscopy Unit (EMU), Medical Faculty, University of Duisburg-Essen, Essen, Germany
• ⁵Department of Physics, School of Natural Sciences, Technical University of Munich, Garching, Germany
• ⁶Munich Institute of Biomedical Engineering, Technical University of Munich, Garching, Germany
• ⁷Division of Neurophysiology, University of Hamburg, Hamburg, Germany

This study identifies and characterizes previously unrecognized medullary B cell niches within murine mucosa-draining lymph nodes (LNs), challenging the conventional understanding of LN architecture. Utilizing advanced imaging techniques, including synchrotron radiation-based phase-contrast micro-computed tomography (SRµCT), correlated high-resolution electron microscopy and immunohistochemistry (IHC), we revealed spherical to ovoid structures termed nodules, being distinct lymphoid compartments consistently localized in the medullary region of mandibular and other mucosa-draining LNs. These nodules were primarily composed of unswitched, non-proliferative CD45R+ B cells expressing IgD and IgM, lacking germinal center features or typical activation markers. They were seamlessly integrated into the medullary architecture, surrounded by LYVE-1+ lymphatic endothelial cells, and situated in close proximity to medullary high endothelial venules (HEVs), revealed by PNAd staining. Under steady-state conditions, this has not been previously observed in the medullary compartment of LNs but is likely facilitating nodule-like B cell aggregation in mucosa-draining LNs due to sustained low level antigenic stimulation common in mucosal environments and is underpinned by proteomics. Additionally, the nodules displayed a capillary network that closely resembles the vascularization seen in conventional B cell follicles revealed by SRµCT. Nodule formation is between two and four weeks postnatally, thus emerging later than B cell follicles, and their abundance significantly increases with age. Functionally, these nodules appear to represent a quiescent B cell niche, potentially supporting B cell homeostasis, tolerance, or memory-like readiness, and are distinct from pathological hyperplasias. Their preservation in aged LNs, coupled with the absence of lipomatosis, suggests a role in maintaining structural integrity and immune readiness through persistent B cell-stromal interactions. This research challenges the established paradigm of LN microarchitecture and suggests specialized niches for B cell function and lymphocyte trafficking in regions subject to constant antigenic exposure.