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

As secondary lymphoid organs, the spleen and lymph node represent important hubs for both innate and adaptive immunity1,2. Neuroanatomical and tracing data, largely derived from rodents, suggest that lymph nodes contain sensory and sympathetic innervation3,4, whereas the spleen contains postganglionic sympathetic innervation5, with conflicting views regarding the existence of cholinergic or vagal innervation6. Herein, we map the neuronal, vascular, and sinus cell networks from human spleen and lymph node using highly multiplexed CODEX (CO-Detection by indEXing) and 3D light sheet microscopy of cleared tissues. These data demonstrate striking delineation of two distinct layers within the lymph node subcapsular sinus--the ceiling defined by Podoplanin expression and floor by LYVE1, which overlays the lymph node follicles. Within the lymph node interior, we observed a mesh-like vessel network innervated with GAP43 and {beta}3-tubulin. Dense perivascular innervation occurred in both tissues, including a subset of axonal processes expressing choline acetyl transferase (ChAT). Four neuronal markers (ChAT, PGP9.5, tyrosine hydroxylase, and {beta}3-tubulin) localized to the arterial tunica externa suggest expression in the nervi vasorum while GAP43 was expressed within the internal elastic membrane of arteries. These data represent highly novel 3D visualization of perivascular and periductal autonomic innervation within these two key human organs.