Immunomodulatory Responses of Subcapsular Sinus Floor Lymphatic Endothelial Cells in Tumor-Draining Lymph Nodes

SIMPLE SUMMARY: Lymph nodes (LNs) are essential for the activation of immune responses against tumors. LNs consist of immune cells and stromal cells, including lymphatic endothelial cells (LECs), that closely interact with each other. We performed single-cell RNA sequencing of LECs residing in tumor-draining LNs compared to normal LNs, to investigate their responses to tumor-derived signals. We found that a specific subset of LECs lining the floor of the subcapsular sinus, where the afferent lymph enters the LN, showed the most drastic changes in gene expression. Many of the upregulated genes were associated with inflammation and vessel growth. Furthermore, we found that several upregulated genes, including podoplanin, mediate adhesion of macrophages to LN LECs. Consequently, deletion of podoplanin on LECs reduced the number of LN macrophages in vivo. Our study shows that tumor-derived signals induce changes in LN LECs that may influence the tumor immune response. ABSTRACT: Tumor-draining lymph nodes (LNs), composed of lymphocytes, antigen-presenting cells, and stromal cells, are highly relevant for tumor immunity and the efficacy of immunotherapies. Lymphatic endothelial cells (LECs) represent an important stromal cell type within LNs, and several distinct subsets of LECs that interact with various immune cells and regulate immune responses have been identified. In this study, we used single-cell RNA sequencing (scRNA-seq) to characterize LECs from LNs draining B16F10 melanomas compared to non-tumor-draining LNs. Several upregulated genes with immune-regulatory potential, especially in LECs lining the subcapsular sinus floor (fLECs), were identified and validated. Interestingly, some of these genes, namely, podoplanin, CD200, and BST2, affected the adhesion of macrophages to LN LECs in vitro. Congruently, lymphatic-specific podoplanin deletion led to a decrease in medullary sinus macrophages in tumor-draining LNs in vivo. In summary, our data show that tumor-derived factors induce transcriptional changes in LECs of the draining LNs, especially the fLECs, and that these changes may affect tumor immunity. We also identified a new function of podoplanin, which is expressed on all LECs, in mediating macrophage adhesion to LECs and their correct localization in LN sinuses.