The lymphatic sinuses in human lymph nodes (LNs) are crucial to LN function yet their structure remains poorly defined. Much of our current knowledge of lymphatic sinuses derives from rodent models, however human LNs differ substantially in their sinus structure, most notably due to the presence of trabeculae and trabecular lymphatic sinuses that rodent LNs lack. Lymphatic sinuses are bounded and traversed by lymphatic endothelial cells (LECs). A better understanding of LECs in human LNs is likely to improve our understanding of the regulation of cell trafficking within LNs, now an important therapeutic target, as well as disease processes that involve lymphatic sinuses. We therefore sought to map all the LECs within human LNs using multicolor immunofluorescence microscopy to visualize the distribution of a range of putative markers. PROX1 was the only marker that uniquely identified the LECs lining and traversing all the sinuses in human LNs. In contrast, LYVE1 and STAB2 were only expressed by LECs in the paracortical and medullary sinuses in the vast majority of LNs studied, whilst the subcapsular and trabecular sinuses lacked these molecules. These data highlight the existence of at least two distinctive populations of LECs within human LNs. Of the other LEC markers, we confirmed VEGFR3 was not specific for LECs, and CD144 and CD31 stained both LECs and blood vascular endothelial cells (BECs); in contrast, CD59 and CD105 stained BECs but not LECs. We also showed that antigen-presenting cells (APCs) in the sinuses could be clearly distinguished from LECs by their expression of CD169, and their lack of expression of PROX1 and STAB2, or endothelial markers such as CD144. However, both LECs and sinus APCs were stained with DCN46, an antibody commonly used to detect CD209.

Lymph nodes (LNs) form the intersection between the vascular and lymphatic systems. Lymphocytes and antigen-presenting cells (APCs) traffic between these systems, but the barriers crossed during this trafficking in human LNs are poorly defined. We identified a population of cells in human LNs that lines the boundary between the parenchyma and lymphatic sinuses, consistent with descriptions of marginal reticular cells (MRCs) in murine LNs. Human MRCs are CD141high podoplanin + , CD90 + , ICAM1 + , and VCAM1 + but lack endothelial and hematopoietic cell markers, or alpha-smooth muscle actin. We then examined expression of the enzyme sphingosine-1-phosphate (S1P) lyase (SGPL1) relative to the boundary defined by MRCs. SGPL1 expression was almost exclusively restricted to cells on the parenchymal side of MRCs, consistent with a role in maintaining the S1P gradient between the sinuses and the parenchyma. Surprisingly the cells expressing SGPL1 in the parenchyma were CD68 + APCs. CD68 + APCs generated from human monocytes were able to internalize and irreversibly degrade S1P, and this activity was inhibited by the S1P analogue FTY720. This work provides a map of the key structures at the boundary where human lymphocytes egress into sinuses, and identifies a novel potential mechanism for the activity of S1P analogues in humans.Keywords: FTY720 r Lymph node r Lymphocyte egress r Marginal reticular cells r Sphingosine 1-phosphate Additional supporting information may be found in the online version of this article at the publisher's web-site IntroductionLymph nodes (LNs) provide an interface between the blood and lymphatic systems, enabling presentation of antigen from Armed with the ability to identify MRCs, we were able to examine the boundaries where T-lymphocytes cross from parenchymal areas into lymphatic sinuses, and study some of the cells and molecules that control this egress. Trafficking into sinuses, which is crucial to the recirculation of lymphocytes, has been observed at both paracortical and medullary sites in rodent models [3][4][5]. In humans, lymphocyte egress into the sinuses is now a validated therapeutic target, with the advent into the clinic of the egressblocking immunosuppressive drug FTY720 (Fingolimod).Rodent models have demonstrated that sphingosine-1-phosphate (S1P) is involved in regulating lymphocyte egress into lymphatic sinuses [6][7][8][9]. S1P acts as a chemoattractant for lymphocytes expressing S1P receptors [6,10]. In murine LNs, lymphatic endothelial cells (LECs) secrete S1P [11], and there is strong evidence that a gradient of S1P is generated across the boundary between the parenchyma and the sinuses, dependent on the function of S1P lyase (SGPL1) [10]. Inhibition, knock down, or knock out of SGPL1 results in elevated levels of S1P in the parenchyma and sequestering of lymphocytes in the LNs [10,[12][13][14]. However, the primary cell types that mediate S1P degradation by SGPL1 have not previously been identified in either rodent or human LNs [15]. FTY720, once phosphorylated in the ...

◥Metastasis of human tumors to lymph nodes (LN) is a universally negative prognostic factor. LN stromal cells (SC) play a crucial role in enabling T-cell responses, and because tumor metastases modulate their structure and function, this interaction may suppress immune responses to tumor antigens. The SC subpopulations that respond to infiltration of malignant cells into human LNs have not been defined. Here, we identify distinctive subpopulations of CD90 þ SCs present in melanoma-infiltrated LNs and compare them with their counterparts in normal LNs. The first populationcorresponds to fibroblastic reticular cells that express various T-cell modulating cytokines, chemokines, and adhesion molecules. The secondSCs embedded in collagenous structures, such as the capsule and trabeculae, that predominantly produce extracellular matrix. We also demonstrated that these two SC subpopulations are distinct from two subsets of human LN pericytes, CD90 þ CD146 þ CD36 þ NG2 À pericytes in the walls of high endothelial venules and other small vessels, and CD90 þ CD146 þ NG2 þ CD36 À pericytes in the walls of larger vessels. Distinguishing between these CD90 þ SC subpopulations in human LNs allows for further study of their respective impact on T-cell responses to tumor antigens and clinical outcomes.