Antiangiogenic treatment targeting the vascular endothelial growth factor (VEGF) signaling pathway is in clinical use, but its effect on vascular function and the tumor microenvironment is poorly understood. Here, we investigate cross‐talk between VEGF and proinflammatory TNF‐α signaling in endothelial cells and its impact on leukocyte recruitment. We found that cotreatment with VEGF decreased TNF‐α‐induced Jurkat cell adhesion to human microvascular endothelial cells by 40%. This was associated with inhibition of TNF‐α‐mediated regulation of 86 genes, including 2 T‐lymphocyte‐attracting chemokines, CXCL10 and CXCL11 [TNF‐α concentration 1 ng/ml; 50% inhibition/inhibitory concentration (IC ₅₀ ) VEGF, 3 ng/ml]. Notably, VEGF directly suppressed TNF‐α‐induced gene expression through negative cross‐talk with the NF‐κB‐signaling pathway, leading to an early decrease in IFN regulatory factor 1 (IRF‐1) expression and reduced phosphorylation of signal transducer and activator of transcription 1 (p‐Stat1) at later times. Inhibition of VEGF signaling in B16 melanoma tumor‐bearing mice by sunitinib treatment resulted in up‐regulation of CXCL10 and CXCL11 in tumor vessels, accompanied by up to 18‐fold increased infiltration of CD3 ⁺ T‐lymphocytes in B16 tumors. Our results demonstrate a novel role of VEGF in negative regulation of NF‐κB signaling and endothelial activation in the tumor microenvironment and provide evidence that pharmacological inhibition of VEGF signaling enhances T‐lymphocyte recruitment through up‐regulation of chemokines CXCL10 and CXCL11.—Huang, H., Langenkamp, E., Georganaki, M., Loskog, A., Fuchs, P. F., Dieterich, L. C., Kreuger, J., Dimberg, A., VEGF suppresses T‐lymphocyte infiltration in the tumor microenvironment through inhibition of NF‐κB‐induced endothelial activation. FASEB J. 29, 227–238 (2015). www.fasebj.org

The lymphatic system, composed of initial and collecting lymphatic vessels as well as lymph nodes that are present in almost every tissue of the human body, acts as an essential transport system for fluids, biomolecules, and cells between peripheral tissues and the central circulation. Consequently, it is required for normal body physiology but is also involved in the pathogenesis of various diseases, most notably cancer. The important role of tumor-associated lymphatic vessels and lymphangiogenesis in the formation of lymph node metastasis has been elucidated during the last two decades, whereas the underlying mechanisms and the relation between lymphatic and peripheral organ dissemination of cancer cells are incompletely understood. Lymphatic vessels are also important for tumor-host communication, relaying molecular information from a primary or metastatic tumor to regional lymph nodes and the circulatory system. Beyond antigen transport, lymphatic endothelial cells, particularly those residing in lymph node sinuses, have recently been recognized as direct regulators of tumor immunity and immunotherapy responsiveness, presenting tumor antigens and expressing several immune-modulatory signals including PD-L1. In this review, we summarize recent discoveries in this rapidly evolving field and highlight strategies and challenges of therapeutic targeting of lymphatic vessels or specific lymphatic functions in cancer patients.

Lymph nodes (LNs) are highly organized secondary lymphoid organs that mediate adaptive immune responses to antigens delivered via afferent lymphatic vessels. Lymphatic endothelial cells (LECs) line intranodal lymphatic sinuses and organize lymph and antigen distribution. LECs also directly regulate T cells, mediating peripheral tolerance to self-antigens, and play a major role in many diseases, including cancer metastasis. However, little is known about the phenotypic and functional heterogeneity of LN LECs. Using single-cell RNA sequencing, we comprehensively defined the transcriptome of LECs in murine skin-draining LNs and identified new markers and functions of distinct LEC subpopulations. We found that LECs residing in the subcapsular sinus (SCS) have an unanticipated function in scavenging of modified low-density lipoprotein (LDL) and also identified a specific cortical LEC subtype implicated in rapid lymphocyte egress from LNs. Our data provide new, to our knowledge, insights into the diversity of LECs in murine LNs and a rich resource for future studies into the regulation of immune responses by LN LECs.

Tumor-associated lymphatic vessels play multiple roles during tumor progression, including promotion of metastasis and regulation of anti-tumor immune responses by delivering antigen from the tumor bed to draining lymph nodes. Under steady-state conditions, lymph node resident lymphatic endothelial cells have been found to maintain peripheral tolerance by directly inhibiting autoreactive T-cells. Similarly, tumor-associated lymphatic endothelium has been suggested to reduce anti-tumor T-cell responses, but the mechanisms that mediate this effect have not been clarified. Using two distinct experimental tumor models, we found that tumor-associated lymphatic vessels gain expression of the T-cell inhibitory molecule PDL1, similar to lymph node resident lymphatic endothelial cells, whereas tumor-associated blood vessels downregulate PDL1. The observed lymphatic upregulation of PDL1 was likely due to IFN-g released by stromal cells in the tumor microenvironment. Furthermore, we found that blocking PDL1 results in increased T-cell stimulation by antigen-presenting lymphatic endothelial cells in vitro. Taken together, our data suggest that peripheral, tumor-associated lymphatic endothelium contributes to T-cell inhibition, by a mechanism similar to peripheral tolerance maintenance described for lymph node resident lymphatic endothelial cells. These findings may have clinical implications for cancer therapy, as lymphatic expression of PDL1 could represent a new biomarker to select patients for immunotherapy with PD1 or PDL1 inhibitors.