Stromal components and the control of lymphocyte trafficking in human lymph nodes

Abstract

Lymph nodes (LNs) are located at the interface between the blood and lymphatic systems, enabling the presentation of tissue-derived antigen to blood-derived lymphocytes. Understanding immune responses in human LNs requires better knowledge of the stromal cells and structural components of the LN with which immune cells interact. We sought to characterise the stromal components of human LNs and examine the factors controlling lymphocyte migration within human LNs. This study for the first time reports the presence of two new stromal cell populations in human LNs − marginal reticular cells (MRCs) and LN-derived mesenchymal progenitor cells (LN-MPCs). Human MRCs are characterised by their expression of high levels of CD141 (Thrombomodulin) and their unique distribution at the boundary between the parenchyma and lymphatic sinuses in both the T and B cell areas. LN-MPCs mainly reside in the LN capsule and trabeculae, and are characterised by their expression of the haematopoietic and mesenchymal progenitor cell marker CD34 and the stromal marker CD90, and by their ability to differentiate into adipocytes in vitro. We have also established marker panels to unequivocally differentiate the major populations of LN stromal cells by flow cytometry and immunofluorescence microscopy. This enabled us to characterise the relative abundance and localisation of MRCs and LN-MPCs as well as the other key stromal populations including fibroblastic reticular cells (FRCs), follicular dendritic cells (FDCs), blood endothelial cells (BECs), and lymphatic endothelial cells (LECs). Our results provide a molecular map of the stromal cell populations in human LNs and demonstrate that their composition is more complex than previously appreciated. Having explored the distinct stromal components of human LNs, we subsequently examined the factors regulating T cell egress from human LNs. Murine models have demonstrated that T cells leave the LN by migrating into the lymphatic sinuses in response to a gradient of sphingosine-1- phosphate (S1P) and that the function of S1P lyase (SGPL1) is critical in maintaining this gradient. However, the cells that degrade S1P by SGPL1 have not been identified in either murine or human LNs. Our data for the first time demonstrate that CD68+ antigen presenting cells (APCs) in the T cellrich parenchyma of human LNs are the main cell population expressing SGPL1. In vitro-derived CD68+SGPL1+ APCs were able to internalise and irreversibly degrade S1P, and this activity was inhibited by the egress-blocking immunosuppressive drug FTY720. These results suggest that the CD68+SGPL1+ APCs in the parenchyma may control T cell egress from human LNs, and identify a possible cellular target for FTY720. Lastly, we sought to investigate the components regulating B cell trafficking in human LNs. Our results demonstrate that the vascular structures mediating the migration of B cells in and out of human LNs are located in close proximity to the follicles. Our results strongly suggest that B cell migration via the T cell zones may not be necessary, although plasma cells may migrate deep into the T cell zones, consistent with them taking up residence in tracts close to the medullary region. We have also assessed the profile of chemokine receptors expressed by human LN B cells, and examined how the egress of B cells from human LNs is likely to be controlled. Our data identify CD68+ APCs as the main cell type expressing SGPL1 in the follicles of human LNs, suggesting that these APCs are likely to maintain the follicles as an S1P-low environment. Collectively, the results presented in this study improve our understanding of the complex organisation of human LNs, and will enable new studies of the physiology and pathology of human LNs. Our data also provide insight into some of the mechanisms of lymphocyte trafficking in human LNs, and will inform development of new therapeutic strategies targeting those mechanisms.