While adoptive T cell therapies (e.g., anti-CD19 chimeric antigen receptor (CAR)-T cells) have demonstrated remarkable outcomes in patients with leukemias and lymphomas, significant variability remains in the potency and durability of the antitumor response, and their success against solid tumors has been limited. Previous studies have identified several key determinants of therapeutic efficacy, including distinct T-cell subpopulations in the CAR-T cell infusion product. CAR-T cell production generally requires ex vivo T-cell activation and expansion, and critical attributes of the CAR-T cell infusion product, including its proliferative capacity, persistence, and antitumor potency, are widely determined during this process. Significant research over the past two decades has established that extracellular matrix (ECM) elasticity, or stiffness, impacts many fundamental cell processes, and impacts various aspects of T cell biology (e.g., synapse formation). However, tissues and ECMs are not linearly elastic materials. The ECM is viscoelastic, with its response to mechanical loading being time dependent. Strong effects of matrix viscoelasticity on stem cell differentiation have been demonstrated, but the interplay of matrix stiffness and viscoelasticity on T cell activation is unknown. This project addresses the hypothesis that matrix viscoelasticity and stiffness during activation will directly impact T cell phenotype and therapeutic efficacy. This hypothesis will be explored via the following specific aims: (1) Assess the effects of matrix viscoelasticity and stiffness on T cell phenotype using ECM mimetic hydrogels with tunable stiffness and viscoelasticity, (2) Explore the mechanism by which matrix mechanics regulate T cell differentiation during activation, and its relation to T cells isolated from patients using scRNA-seq analysis and focusing on the AP-1 pathway, and (3) Elucidate the functional effects of changes in T cell state induced by matrix mechanics both in vitro and in vivo using adoptive transfer studies. Completion of these studies will provide fundamental knowledge regarding the role of matrix mechanics on T cell phenotype and function, with a potential impact on approaches to manufacture T cells for adoptive therapies.

Therapeutic T cells can be used to treat cancer, but it is currently not known how the physical properties of the environment in which T cells are manufactured impacts their function. These studies will address this fundamental question using a combination of cell culture studies, and animal validation studies. The resulting knowledge may alter how T cells are currently manufactured for cancer immunotherapy, and could identify new targets to improve anti- cancer immune responses.