Mapping the thymic neuronal connectome using rabies virus retrograde and anterograde tracers

Abstract Immune organ innervation by the peripheral nervous system enables autonomic function in the development and maintenance of immunity. Specifically, the role of thymic innervation in health and following neurological insults is unknown. An in depth identification of the connectome within the thymus and the extent to which nervous impulses delivered to the thymus affect T cell development and other biological processes within the thymus is needed. We sought to determine the exact nature of innervation within the thymus. Using both flow cytometry and confocal microscopy, we determined that the thymus contains both extrinsic neuronal processes (cell bodies outside of the thymus), and surprisingly, intrinsic neurons (cells bodies within the thymus). We further verified that subsets of neurons within the thymus express tyrosine hydroxylase, BIII tubulin, and/or NeuN, and are distinct from epithelial cells (EPCAM+, UEA1+, Ly51+) and tuft cells (DLCK1+). Furthermore, neuronal cell bodies found within the thymus did not express AIRE, consistent with their identity as neurons. Subsets of intrinsic neurons within the thymus express MHCII implying a potential role in T cell development. Importantly, using rabies virus retrograde and anterograde neurotracers, we mapped a novel, previously undescribed, connectome of neurons within the thymus. Finally, we determined that both epithelial cells and neurons are significantly reduced in RAG deficient mice, indicating a possible co-regulation between thymic neurons and epithelial cells. In summary, we describe a novel network of neurons within the thymus and put forward implications of the thymic connectome’s role in T cell development and establishment of thymic architecture.