The promise of adoptive T-cell therapies—immunotherapy in which T cells are collected from a patient, enhanced, and reinfused—is growing, especially against blood cancers. However, a better understanding is needed of how T cells’ traits and functions are shaped by the mechanical resistance of the tissues they encounter while infiltrating them. The mechanical features of tissues can vary widely, and pathological tissues such as tumor masses or fibrotic tissues are significantly different from healthy tissues.

Now, a research team at the Wyss Institute for Biologically Inspired Engineering at Harvard University and Harvard John A. Paulson School of Engineering and Applied Sciences (SEAS), took a novel biomaterials approach to investigate the effect of tissue mechanics on the state of T cells by engineering a 3D model of the extracellular matrix (ECM). In doing so, they demonstrated a distinct impact of tissue viscoelasticity on T-cell development and function in vitro and in vivo, and identified a molecular pathway driving the phenomenon.

This study, noted Donald Ingber, MD, PhD, Wyss founding director, merges three seemingly disparate fields, biomaterials, immunotherapy, and mechanobiology, to develop an entirely new form of biomaterials-based mechanotherapeutic.

This work is published in Nature Biomedical Engineering in the paper, “Generation of functionally distinct T-cell populations by altering the viscoelasticity of their extracellular matrix.

“Our study provides a conceptual basis for future strategies aiming to create functionally distinct T-cell populations for adoptive therapies by selectively tuning mechanical input provided by biomaterials-based engineered cell culture systems,” noted David Mooney, PhD, Wyss core faculty member.

The team’s engineering of a tunable ECM model, in which they focused on a type of collagen that they found to be key to dictating the mechanical behavior of different tissues, was critical. To mimic natural collagen-based ECM, the team fabricated hydrogels whose stiffness they could tune by varying the concentration of collagen molecules: fewer numbers of collagen molecules produced lower stiffness and higher numbers, higher stiffness. Independently, viscoelasticity became tunable by varying the amounts of a synthetic cross-linker molecule that further networked the collagen molecules. More highly cross-linked collagen molecules produced more elastic hydrogels. The resulting ECM-mimicking hydrogels equally allowed the attachment of pre-activated T cells but, importantly, enabled their stimulation with specific mechanical signals.

“To our knowledge, this is the first ECM model that allows researchers to study T cells with stiffness from viscoelasticity decoupled, and thus enables us and others in the future to investigate how immune and other cells might be mechanically regulated,” said Yutong Liu, PhD, a graduate student in Mooney’s group.

The team performed an extensive analysis of T cells exposed to different viscoelastic conditions. “T cells that experienced a more elastic collagen matrix were more likely to develop into ‘effector-like T cells,’ whereas T cells that experienced a more viscous ECM matrix rather became ‘memory-like T cells,’” said Kwasi Adu-Berchie, PhD, who completed his PhD in Mooney’s lab and is currently a translational immunotherapy scientist at the Wyss Institute. “Importantly, we found that a T cell’s state, resulting from the viscoelasticity of a matrix, even more so from more elastic, less viscous hydrogels, becomes long-term imprinted, as the cell retains a memory of that specific matrix after being transferred to a different one. This could have broad implications for future cell manufacturing.”

Gene expression analysis led the team to the activity of the transcription factor AP-1 that links T cells’ reception of a more elastic, less viscous mechanical environment to a more effector-like gene expression program. The number of AP-1 complexes with specific compositions was increased, and genes depending on them for their expression were enriched, not only in T cells isolated from more elastic hydrogels, but also in T cells isolated from patients’ cancer and fibrotic tissues, which are stiffer and more elastic than neighboring healthy tissues. When they inhibited one of AP-1’s components with a drug, the effects of a more elastic collagen matrix on T cells were prevented.

To investigate how different mechanical stimulations and T cells’ predicted gene expression signatures translated into traits and functions, the team used therapeutic CAR-T cells engineered to bind a specific antigen of a human lymphoma cell line. CAR-T cells that were stimulated in a more elastic collagen matrix in vitro exhibited a stronger ability to kill lymphoma cells. Also in vivo, CAR-T cells stimulated in a more elastic matrix, and adoptively transferred into mice with the same type of lymphoma, were significantly more capable of reducing tumor burden in the animals and extending their lives than CAR-T cells exposed to a less elastic matrix.

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