Peyer’s patches (PPs) are secondary lymphoid organs that monitor the intestinal lumen and initiate adaptive immune responses against pathogens. The efferent lymphatic vessels of PPs serve as the primary pathway for lymphocyte egress from the gut, however little is known about their molecular identity and development.

We performed scRNA-seq of mouse intestinal lymphatic endothelial cells (LECs) from gut and PP samples and used a combination of bioinformatic analysis, immunofluorescence and multiparameter flow cytometry to characterize the different gut LEC populations. We also utilized 3D imaging to examine steps of PP lymphatic development and exploited a nanoindentation technique to provide a map of tissue stiffness across the PP.

PPs harbor a molecularly distinct population of LECs with upregulated gene expression programs related to cell adhesion, complement system, lipid metabolism processes and leukocyte migration. We found four subtypes characterized by unique molecular signatures and spatial distributions, which exhibit both similarities to and differences from previously described lymph node LECs. We provide a three-stage model of PP lymphatic formation during postnatal development, which includes an initial phase of VEGF-C driven lymphatic expansion, followed by VEGF-C independent steps of remodeling and specialization. Furthermore, PPs feature a spatial gradient in stiffness, attributed to the presence of diverse immune cell types. We also highlight how these differences emerge during development due to the presence of various mechanical cues, which coincide with PP lymphatic remodeling.

Our findings identify distinct subtypes PP-specific LECs with an insight into subset-specific marker genes, location and functions. We propose a role of mechanical forces in PP postnatal lymphatic remodeling and specialization. We aim to provide a better understanding on the roles of PP lymphatics in the promotion of beneficial mucosal and systemic immune surveillance during postnatal development and in pathological conditions.