Segmental endothelial cell differentiation – a determinant of T cell trafficking in cancer and viral infections

Abstract

During inflammation, immune cells must rapidly migrate to affected tissues to exert their effector functions. To do so quickly, they utilize the intricate vascular network that interconnects every tissue in our body. The vessels that make up the vascular tree are lined by a heterogenous monolayer of blood endothelial cells (ECs) that regulate various physiological functions necessary for survival. In most settings, leukocyte extravasation is restricted to a particular segment of the vasculature, namely post-capillary venules. This segment is lined by venular ECs (VECs) that are uniquely capable of supporting leukocyte adhesion and extravasation into the surrounding tissue. Here, we explore how CD8+ T cells, an essential immune component in the response against cancer and intracellular pathogens, interact with VECs in various settings. In Chapter 2, we elucidate the contributions of VECs in cancer and show that in addition to healthy tissues, they also mediate T cell extravasation into malignant tissues. In Chapter 3, we find that in settings of acute viral infection, while T cell adhesion is initiated on VECs, some T cells do not extravasate and instead crawl against the direction of blood flow into arterioles, which do not support leukocyte extravasation. In the tumor microenvironment (TME), pro-angiogenic signals prevent the proper differentiation of ECs, resulting in the macroscopic loss of vascular segmentation. However, single-cell RNA sequencing (scRNAseq) revealed that some tumor ECs (tECs) still acquire a venular transcriptional profile. Here, using atypical chemokine receptor 1 (ACKR1) as a venular marker, we show that the TME maintains ACKR1+ VECs that can support T cell extravasation. Accordingly, tumors with more VECs tend to have a greater abundance of infiltrating T cells. This is of particular importance because, across several cancer indications, increased T cell infiltration can be predictive of responsiveness to various immunotherapeutic approaches, such as immune checkpoint blockade (ICB). Phenotypically, intratumoral VECs acquire an immature phenotype that downregulates key adhesion molecules such as intercellular adhesion molecule 1 (ICAM-1) and P-selectin, while marginal VECs have an activated phenotype with high expression of these adhesion molecules and are in close proximity to infiltrating T cells. Thus, our study is the first to show that VEC abundance and maturity are prerequisites for efficient T cell infiltration into tumors. Surprisingly, in acute viral infections, after interacting with VECs, some T cells do not transmigrate into the surrounding tissue and instead crawl against the direction of blood flow. To analyze this rheotactic migratory behavior, we developed an interactive web application that streamlines cell track exploration, quantification, and analysis. The Vector-guided Cell Track Analyzer (VeCTA) application enables the alignment of cell tracks with coordinate axes to facilitate directionality analysis in samples restricted by physical barriers, such as the vascular walls. We use this tool to analyze data acquired from intravital 2-photon excitation microscopy and in vitro flow-chamber assays to show that effector CD4+ and CD8+ T-cells preferentially migrate against the direction of fluid flow, often accumulating in arterioles that do not support T cell extravasation.