Inferring dynamic extracellular matrix composition of the thymus: towards a biomimetic scaffold for T cell culture

Abstract

Immunotherapy is a pioneering approach using T lymphocytes (T cells) to fight cancer and immune deficiency, which together affect millions of patients in the US. Current clinical therapies require patient- or donor-derived T cells, leading to manufacturing delays and limited supply. While stem cell-derived T cells offer a scalable strategy to meet growing demand and increase accessibility to therapy, generating mature and clinically usable T cells through this method is challenging and cell yields remain low. In the body, T cell maturation relies on cell migration through thymic structures that change chemically and physically over time; the extracellular matrix (ECM) has a role in shaping these evolving niches. Existing solutions to producing “off the shelf” engineered T cells use mouse thymic epithelial cell lines and native ECM to adapt key in vivo components of the T differentiation process. However, these solutions are not fully synthetic, limiting their scalability and potential therapeutic use. Additionally, these models of the thymic ECM overlook developmental shifts in thymic structure that might advance an understanding of T cell maturation. Therefore, dynamic biological and chemical properties in the thymic microenvironment are important in efficiently producing mature T cells in vivo. This project constructs a proof-of- concept biomimetic platform for immune cell differentiation from stem cells based on these changing characteristics of the thymic extracellular matrix. Using single-cell RNA sequencing data to quantify gene expression levels, the extracellular matrix composition of the thymus at different developmental stages is computationally characterized, and characteristic ratios of key extracellular matrix components (fibronectin, collagen, and laminin) are identified at these different stages. These ratios are then used to engineer a stage-specific alginate-based hydrogel for cell culture, designed to replicate thymic tissue stiffness and viscoelasticity with tunable properties that reflect different developmental stages. Using this stage-specific platform, researchers can investigate how T cell differentiation, activation, and toxicity are influenced by the thymus's developmental stage. Additionally, this platform could allow for the identification of the optimal stage of thymic development to mimic in T cell culture scaffolds, enabling more scalable T cell expansion.Engineering Sciences S