Biomechanical regulation of T cells: The cytoskeleton at the nexus of force and function

Abstract

The adaptive immune response is a sophisticated and multi-pronged defense mechanism that provides specific and long-lasting protection against infections and cancer. Central to this response are T lymphocytes - immune cells that orchestrate the immune response and directly eliminate infected or malignant cells. T cell function is intricately linked to their cytoskeleton, a dynamic network of protein filaments, consisting of actin, microtubules, and intermediate filaments, which provides structure, facilitates movement, and regulates intracellular transport. While the biochemical aspects of T cell function have been well-studied, recent advances have highlighted how mechanical forces influence T cell behaviors such as activation, migration, and effector functions—all processes driven by dynamic cytoskeletal remodeling. However, the mechanisms by which cytoskeletal dynamics, forces and mechanical stimuli drive T cell function remain poorly understood. This dissertation investigates this interplay, focusing on cytotoxic T lymphocytes (CTLs), a subtype of T cells that directly kill infected or cancerous cells. To launch a killing response, naïve CD8+ T cells must be activated by antigen-presenting cells (APCs) in lymph nodes, following which they proliferate and differentiate into an effector CTL population. CTLs eliminate targets via a specialized interface called the immunological synapse (IS), where they release lytic granules containing cytotoxic molecules and exert cytoskeletal forces to induce target cell death. A key event in IS formation is polarization of the centrosome, or the microtubule-organizing center, facilitating directional release of lytic granules. We first examined how biochemical signals provided by APCs modulate the cellular cytoskeleton. APCs provide not only antigenic stimulation, but also co-stimulatory signals required for full activation. Inflammatory cytokines such as interleukin-12 (IL-12) act as a third signal, enhancing CTL proliferation and cytotoxicity. Our findings demonstrate that CTLs activated in the presence of IL-12 exhibit enhanced IS formation, altered actin dynamics and microtubule growth, and generate greater mechanical forces, thus highlighting how activation signals can shape T cell mechanics, dynamics and function. Next, we investigated how the mechanical properties of target cells influence CTL function. Employing a biomimetic hydrogel system that mimics the stiffness of target cells, we demonstrate that substrate stiffness modulates multiple aspects of CTL responses. CTLs interacting with stiffer substrates exhibit enhanced spreading, accelerated actin ring formation, increased contractile forces, and more efficient centrosome polarization. Mechanical cues also influence lytic granule release and the nuclear translocation of mechanosensitive transcription factors. This work underscores the importance of mechanical cues in regulating immune responses. Given that coordinated cytoskeletal interactions are crucial for T cells to effectively respond to environmental cues, we further examined this crosstalk with a focus on intermediate filaments, the third, often understudied component of the cytoskeleton. Our characterization of the vimentin intermediate filament network reveals an expansive structure complementary to and dependent on other cytoskeletal components. We study the dynamics and organization of the vimentin network and find a close association of this network with the centrosome. Our results suggest a structural role for vimentin in supporting IS formation. Throughout this work, we use advanced imaging techniques and analysis approaches to probe various facets of T cell function. By bridging immunology, cell biology, and biophysics, this research contributes to our understanding of how physical forces shape immune responses.