Cellular landscape of reactive and neoplastic human lymph nodes in 3D

Lymph nodes function according to cellular and structural regulations. When these rules deviate from the benign equilibrium, dysregulations occur leading to the onset of diseases. In the development of malignant lymph node diseases, processes can be observed that consistently follow the same pattern. This study aims to define the structural and cellular parameters of the reactive lymph node and to demonstrate how lymph nodes change during tumorigenesis. We analysed benign cases diagnosed as Lymphadenitis (8 patients). Malignant cases with the diagnosis of Nodular Sclerosis classical Hodgkin Lymphoma (5 patients), Mixed Cellularity classical Hodgkin Lymphoma (5 patients), Follicular Lymphoma (7 patients), and diffuse large B-cell Lymphoma (2 patients) were selected. Confocal microscopy was used to visualise immune cells and their compartments in 3D. Based on the fibroblastic reticular cell network we defined five compartments in reactive lymph nodes: Subcapsular sinus, marginal sinus, follicle, T-zone, and medulla. We analysed the cellular composition based on dendritic cells, macrophages, T cells and B cells and extended this analysis to include the presence of extracellular vesicles. During tumorigenesis, the compartmentalisation of the lymph node is progressively destroyed. Despite this ongoing destruction and loss of strict compartmental delineations, at least two distinct structural regions are still visible, defined as follicle-like and T-zone-like compartments. A comparison between reactive and neoplastic cases reveals a progressive cellular and structural homogenisation. Higher masses of CD8⁺ T cells were found under neoplastic conditions and higher masses of CD30⁺ were found in Hodgkin Lymphoma. The volume of CD20⁺ cells in follicles was consistently lower in malignant tissues compared to benign, but higher in T-zones in malignant cases. An increase in vesicles was detected in most neoplasms. These findings offer new insights into cellular and structural remodelling, deepening our understanding of tumorigenesis and paving the way for more precise therapeutic interventions.