Study on T cell dynamics in complex microenvironments using microfluidic systems

Abstract

T cell extravasation and tissue infiltration are critical steps in antigen-specific immune responses in peripheral tissues to repair tissue injury and to fight against infections and cancers. T cells activated by antigen presenting cells (e.g. dendritic cells) in lymph nodes enters blood circulation to efficiently traffic to inflamed tissues. T cell extravasation occurs through sequential dynamic interactions between T cells and with components in inflamed blood vessels, known as a leukocyte adhesion cascade. The leukocyte adhesion cascade is composed of rolling, arrest and firm adhesion, intraluminal crawling (ILC), trans-migrate through ECs (transendothelial migration, TEM), subendothelial crawling (SEC) to bleach basement membrane. After performing extravasation, T cells crawl interstitial spaces filled with extracellular matrixes and cells by amoeboid-like motility and eventually arrive at the target sites where they will perform effector functions. Both extravasation and interstitial migration, dynamics of T cells is regulated by various biochemical and biophysical signals in complex microenvironments near blood vessels. In this thesis, we sought to investigate the roles of complex microenvironments in regulating T cells dynamics by reconstructing various features of in vivo microenvironments into in vitro microfluidic systems. Biochemical/biophysical factors regulating each step of leukocyte adhesion cascade have been extensively studied . However, how EC adhesion affects leukocyte extravasation, in particular TEM and SEC, has not been fully understood. In Chapter II, we employed interference reflection microscopy (IRM), a microscopy technique specialized for label-free visualization of cell–substrate contact, to study detailed dynamic interactions between basal part of ECs and T cells underneath EC monolayer. For TEM, T cells on EC monolayer extended protrusions through junctions to explore subendothelial spaces, and EC focal adhesions (EC-FAs) acted as physical barrier for the protrusion. Therefore, preferential TEM occurred through junctions where near-junction focal adhesion (NJ-FA) density of ECs was low. After TEM, T cells performed subendothelial crawling (SEC) with flattened morphology and reduced migration velocity due to tight confinement. T cell SEC mostly occurred through gaps formed in between EC-FAs with minimally breaking EC-FAs. Tumor necrosis factor- (TNF-) treatment significantly loosened confinement in subendothelial spaces and reduced NJ-FA density of ECs, thus remodelled basal part of EC layer to facilitate leukocyte extravasation. Artery stenosis is a key feature of atherosclerosis that causes serious cardiovascular diseases. The development and progression of atherosclerosis are influenced by T cells recruited from the blood through the secretion of various pro-inflammatory cytokines. The formation of stenosis disturbs flow that can affect T cell infiltration in the arterial walls. However, the influence of complex flow environments on the dynamic behaviors of T cells in blood vessels has yet to be investigated. In Chapter III, stenotic structures mimicking artery stenosis were fabricated and the dynamic behaviors of T cells on endothelial layers of post-stenotic areas were investigated. The flow characteristics, including wall shear stress, vertical velocity, and axial velocity fluctuations of the stenotic chamber were calculated through a particle image velocimetry (PIV) technique and numerical simulation. T cells were flowed in the stenotic chamber containing endothelial layers at the bottom of the post-stenotic areas. The detailed dynamic behaviors of T cells were observed through video microscopy and then analyzed to examine the roles of complex flow on each step of T cell adhesion cascades for infiltration. T cell infiltration into tumor is a key factor determining efficacy of cancer immunotherapy. In order for activated T cells in blood stream to exert cytotoxicity against solid tumor, they need to undergo three steps, including extravasation to leave blood vessels, interstitial migration to directly contact tumor cells, and cytolytic synapse formation to kill tumor cells. In Chapter IV, we developed a multi-layered blood vessel-tumor tissue chip that allow us to examine aforementioned three steps of T cell tumor infiltration. The multi-layered blood vessel-tumor tissue chip is composed of a top fluidic channel, a porous membrane covered with a confluent EC monolayer, and a 3D collagen gel encapsulating a melanoma cell (MC) monolayer. Using this microfluidic chip, we could directly observe T cells performed extravasation and migrated toward MCs via interstitial migration to eventually kill them using live cell imaging. Interestingly, presence of MCs influenced both extravasation and interstitial migration of T cells. T cell extravasation was significantly impaired on ECs co-cultured with MCs, as ECs co-cultured with MCs failed to express adhesion molecules in response to TNF- stimulation. Unresponsiveness to inflammatory cytokines such as TNF- , or EC anergy, could be reverted by treating EC-MC co-culture with anti-vascular endothelial growth factor A (VEGFA) antibody, confirming angiogenesis signals triggered by tumor cells can cause EC anergy. Substantial fractions of extravasated T cells migrated toward MCs and killed them, whereas in the absence of MCs, the majority of them remained near ECs, suggesting MCs secrete chemo-attracting molecules to attract T cells. T cells pre-treated with pertussis toxin (PTX), an irreversible inhibitor of G-protein coupled receptor (GPCR), did not migrated toward MCs and remained near ECs after extravasation, confirming MC-mediated chemotaxis is major factor for interstitial migration of T cells. This microfluidic chip can be useful not the only for fundamental investigation of tumor-EC-T cell interactions but also for pre-clinical evaluation of ex vivo engineered cytotoxic lymphocytes for cancer immunotherapy.