Development of synnotch platform for sensing receptor force and investigation of receptor-mediated mechanotransduction
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Lyu, Jintian
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Abstract
B cell lymphoma, a malignancy stemming from B lymphocytes, is a dynamic disease marked by the uncontrolled growth of B cells and their accumulation in various organs, presenting a complex health challenge. This dissertation interrogates the multifaceted interplay of genetic mutations that contributes to the pathogenesis and progression of B cell lymphoma, with particular focus on how it shapes mechano-transduction of CD40 in B cell. We explored the germinal center (GC) origin of Diffuse Large B-cell Lymphoma (DLBCL), examining the role of epigenetic modulations and mutations in the EZH2 gene, a histone methyltransferase, which impact B cell functionality and development. Particularly, we consider how EZH2 mutations can initiate lymphomagenesis by altering the dependency of GC B cells on T follicular helper (Tfh) cells. By harnessing the mechano-sensitive properties of the Notch receptor, we have engineered a synthetic version of the Notch receptor (SynNotch) by substituting its native ligand-binding domain with an antibody's single-chain variable fragment, enabling targeted receptor interaction, and modifying its signaling pathway to trigger a detectable response, such as the production of green fluorescence protein (GFP) or luciferase. When a target cell applies force to the receptor, which then gets relayed to the SynNotch, this interaction prompts the signal for GFP or luciferase production, visible through flow cytometry or microscopy. By introducing cells equipped with this SynNotch construct into living organisms, we can trace their journey to locate and engage with cells that express the intended receptor, monitoring the subsequent reporter signals. This approach permits the quantification of the exerted forces on the receptor by the target cells in a living context. We demonstrated the design, in vitro validation, and in vivo application of this system using T cells as sensor cells and B cells as targets, focusing on the CD40 receptor. Our research confirms the practicality and value of our platform, offering a novel method for future mechanobiological studies. Through this, we demonstrate that the EZH2Y641 mutation alters the mechanical interaction between B cells and the CD40-CD40L interface—a critical point in immunoregulation—disrupting the downstream signaling and epigenetics that ordinarily extinguish the pseudo-malignant phenotype of GC B cells. Our results also reveal that the EZH2Y641 mutation reduces in situ CD40–CD40L affinity, a discrepancy that can be corrected by EZH2 inhibition. Further investigations show that mechanical forces influence the phosphorylation of key signaling molecules and can modulate the spreading of B cells on CD40L presenting surfaces, impacting B cell signaling. The utilization of the SynNotch platform to compare the mechanical behavior of B cells in vivo, between those with wild-type EZH2 and the EZH2Y641 mutant, underscores the utility of our technology. Collectively, this dissertation elucidates the mechanobiological mechanisms at play in the malignant transformation of B cells and validates a new technology to study them, thus contributing valuable insights into the underpinnings of B cell lymphomas and establishing a groundwork for potential therapeutic interventions. In the end of the thesis, we extended our investigation into other biological system and show the important role of mechano-transduction.
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2024-04-26
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Dissertation