The mechanism how Pretubulysin-induced microtubule disassembly improves T cell search efficiency

Cell migration is a crucial process for cytotoxic T lymphocytes (CTLs), that allows effective navigation through three-dimensional (3D) environments to locate target cells and execute cytotoxic functions. Impaired CTLs infiltration into solid tumors remains a significant challenge, often limiting the efficacy of immunotherapies. To address this challenge, recent evidence suggests microtubules (MT) as a promising target, since perturbing MT stability improves T-cell migration and killing efficiency in dense matrices, but the underlying mechanisms remain poorly understood. Here we use Pretubulysin, a known MT depolymerizer to unmask mechanism how MT stability influences T-cell migration. We found that complete disassembly of the microtubule network significantly increased CTLs infiltration and migration in a 3D environment. Asking how migration actually improves killing efficiency, we focused on the cell search efficiency, since finding a target is a crucial prerequisite to kill it. Using a persistent random walk model, we prove the ability of Pretubulysin to enhance T-cell search efficiency. We are proposing an underlying mechanism explaining this enhanced searching efficiency of T cells after Pretubulysin treatment: Depolymerizing microtubules in activated T cells leads to localized actomyosin accumulation at the uropod, altering cell mechanics and favoring stronger contraction forces at the rear of the cell. This results in faster and more persistent migration. Overall, our findings shed light on the role of MTs in search mechanisms of immune cells, regulating CTLs migration in 3D environments. We highlight the potential of MT-disassembling agents like Pretubulysin to optimize immune therapies against solid tumors. Significance StatementWe show how microtubule (MT) disruption with Pretubulysin can be used to enhance cell search behavior and therewith the function of CTLs. By inducing rapid and potent depolymerization of MTs, Pretubulysin treatment induces profound changes in CTLs behavior and mechanics. Pretubulysin-treated CTLs exhibit increased migration speed and persistence, leading to improved infiltration, search and therefore killing in complex 3D. These enhancements are explained by alterations in cell stiffness, morphology, and relocalization of cytoskeletal elements, including enrichment of actomyosin activity at the uropod. We provide necessary insights into the intricate interplay between cytoskeletal dynamics and CTLs function. This knowledge will inform the development of novel immunotherapeutic strategies targeting the cytoskeleton to boost T cell-mediated cytotoxicity in the treatment of various diseases.