3D-mapping of human lymph node and spleen reveals integrated neuronal, vascular, and ductal cell networks

The spleen and lymph node represent important hubs for both innate and adaptive immunity1,2. Herein, we map immune, endothelial, and neuronal cell networks within these tissues from "normal"/non-diseased organ donors, collected through the NIH Human BioMolecular Atlas Program (HuBMAP)3, using highly multiplexed CODEX (CO-Detection by indEXing) imaging and 3D light sheet microscopy of cleared tissues. Building on prior reports4-6, we observed the lymph node subcapsular sinus expressing podoplanin, smooth muscle actin, and LYVE1. In the spleen, LYVE1 was expressed by littoral cells lining venous sinusoids, whereas podoplanin was restricted to arteries and trabeculae. 3D visualization of perivascular innervation revealed a subset of axonal processes expressing choline acetyl transferase in both tissues, in contrast with prior literature on human spleen7. We further report our novel observations regarding the distinct localization of GAP43 and {beta}3-tubulin within the vascular anatomy of both lymph node and spleen, with Coronin-1A+ cells forming a dense cluster around {beta}3-tubulin positive GAP43 low/negative segments of large vessels in spleen. These data provide an unprecedented 2D and 3D visualization of cellular networks within secondary lymphoid tissues, laying the groundwork for future disease-specific and system-wide studies of neural regulation of immunity in human lymphatics.