Membrane, phase separation, and cancer immunity

 

The cell membrane not only separates the intracellular space from the external environment, but also provides a platform for the receiving, processing and transduction of extracellular stimuli. The two-dimensional geometry, the interaction between proteins and lipids, and the local membrane curvature all converge to generate emergent properties of membrane-proximal signaling whereas the underlying mechanisms and functional consequences are not well understood. Immune receptor signaling represents a good example of this scenario. The highly dynamic membranes in the synapse and multiple ligand-receptor interaction pairs posted numerous exciting questions to explore in both basic biology and immunotherapy development.

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Our lab has established an interdisciplinary platform to characterize and engineer immune signaling across scales. It consists of membrane-based biochemical reconstitution, live cell imaging of subcellular structures, cell engineering for new functions, and animal models to test in vivo effects. Currently we implemented the above approaches to understand immune signaling in the context of 1) T cells' attacking of cancer cell; 2) mast cells' interaction with surrounding tissues and tumors; 3) Leukocytes' transcellular migration through endothelial cells.

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Mechanism of CAR-T cell activation

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The chimeric antigen receptor (CAR) enables T cells to specifically target and kill cancer cells. Despite of its success in clinical trials, the cellular mechanism of how CAR is activated by antigen and how activated CAR triggers downstream signaling pathways remains unclear. We have established a supported lipid bilayer system coupled with TIRF imaging for visualizing CAR signaling at high spatial and temporal resolutions (paper). These enable my group to comprehensively investigate and manipulate the signaling pathway in both regular and CAR-T cells. We revealed a size-dependent mechanism explaining how antigen engagement triggers CAR activation (paper), which solves a long-standing question in the CAR field. We also discovered that CAR induces an unstable synapse that has a disorganized pattern; moreover, CAR bypasses LAT, a key adaptor in the TCR pathway, to activate T cells (paper). These knowledge in basic immune signaling guided us to design new CARs and engineer immune cells to improve the antitumor responses. Currently we are exploring the following questions:

• How is signaling amplified along the CAR pathway?

• How does phase separation affect CAR signaling?

• How to design CARs with improved antigen sensitivity?

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Mast cell granule and anti-tumor function 

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Mast cells are among the least understood immune cells though they are widely distributed in tissues and communicate with a variety of immune and stroma cells either through direct contacts or secreted mediators. The cytoplasm of mast cells is filled up with granules that contain multiple mediators including bioactive chemicals, proteases, cytotoxic factors, chemokines, signaling lipids and glycans. Interestingly, many of these mediates remain in the granules even after their release into the extracellular space where there are no membranes wrapping around granules. The underlying biochemical mechanism is unclear. Functionally, mast cells were traditionally associated with inflammatory diseases including asthma and urticaria. We propose to repurpose the inflammatory role of mast cells for an anti-tumor function. This is expected to overcome some of the major hurdles preventing T cell-centered therapy for solid tumors. Currently we are exploring the following questions:

• How do mast cell extracellular granules maintain their structural and functional integrity?

• Can we program mast cells to target solid tumors through developing novel CARs?

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Transcellular migration of leukocytes

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During an immune response to pathogen infection, Leukocytes, which normally circulate in the vascular system, transmigrate through the endothelial layer to reach infected tissues and clear pathogens. Similarly, circulating metastatic cancer cells transmigrate through the endothelial layer to reach new colonization sites. In traditional views, transendothelial migration occurs at cell-cell junctions (paracellularly). However, recent evidence suggested the presence of transcellular migration, in which leukocytes or cancer cells penetrate through the endothelial cell to exit blood vessel. This transcellular process requires intimate interactions and bidirectional signaling between the invading and receiving cells, accompanied by highly coordinated remodeling of cytoskeleton and membrane systems. Currently we are exploring the following questions:

• What are the ligand-receptor pairs that mediate bi-directional signaling between endothelial cells and leukocytes?

• How do endothelial cells remodel their membranes to accommodate transcellular migration?

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