T Cell Traits Shaped With Biomechanical Materials

Adoptive T cell treatments, a type of immunotherapy in which immune T cells are taken from a patient, boosted outside of the body, then reinfused back into the same patient, are now being utilized to treat blood malignancies. However, enhancing the ability to generate patient-specific T cell populations with specific features and functions could widen clinicians’ T cell therapy repertoire.

One approach to this goal is to better understand how the mechanical resistance of the tissues they encounter while infiltrating them shapes T cells’ traits and functions, such as their cytotoxic effects on unwanted target cells (effector T cells) or their ability to recall and eliminate them if they reappear (memory T cells). The mechanical properties of tissues, such as bone, muscle, various internal organs, and blood, can differ greatly, and diseased tissues, such as tumor masses or fibrotic tissues, differ mechanically dramatically from healthy tissues.

A research team led by Wyss Core Faculty member David Mooney, Ph.D. at Harvard University’s Wyss Institute for Biologically Inspired Engineering 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.

They were able to control these characteristics independently by building a 3-dimensional model of the extracellular matrix (ECM), which is formed by cells that are responsible for the varied stiffnesses and viscoelasticities of tissues. This allowed them to show that tissue viscoelasticity has a different impact on T cell growth and function in vitro and in vivo, as well as discover a biochemical route underlying the phenomena. Nature Biomedical Engineering published the findings.

Mechanical resistance manifests itself as “stiffness,” a tissue’s (or any material’s) resistance to instantaneous deformation, and “viscoelasticity,” the sort of relaxation that occurs after deformation. In physical words, a viscous (fluid) material, such as honey, is more likely to flow, whereas an elastic (solid) material, such as a rubber band after stretching—and this is true for tissues that contain both solid and fluid components.

“Importantly, the phenotypes, functions, and gene expression programs of T cells trained in variations of the system correlated well with those we found in T cells in mechanically distinct tissues from patients with cancer or fibrosis,” said Mooney who also is the Robert P. Pinkas Family Professor of Bioengineering at SEAS, and leads the Wyss Institute’s Immunomaterials Initiative. “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.”

In a dish, mimicking tissue mechanics

The team’s engineering of an adjustable ECM model, in which they focused on a type of collagen that they discovered to be critical in determining the mechanical behavior of different tissues, was critical to their discoveries. Collagen is a key ECM protein that is secreted by nearly every cell in the body. Individual collagen protein molecules are naturally arranged into crimped fibrils, which then chemically cross-link to form fibers. Each fibril can be thought of as a mechanical spring, and each fiber as a collection of springs. The stiffness of an ECM is determined by how tightly collagen molecules are packed, but its unique viscoelasticity is determined by how densely collagen molecules are cross-linked to each other.

To replicate natural collagen-based ECM, the researchers created hydrogels with adjustable stiffness by altering the concentration of collagen molecules: fewer collagen molecules produced lower stiffness, while more collagen molecules produced higher stiffness. Viscoelasticity was independently made programmable by altering the quantities of a synthetic cross-linker molecule that further networked the collagen molecules. More elastic hydrogels were formed when collagen molecules were heavily cross-linked. The resulting ECM-mimicking hydrogels allowed pre-activated T cells to attach but, more critically, allowed them to be stimulated with particular mechanical cues.

“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 co-first author Yutong Liu, Ph.D., who was a graduate student in Mooney’s group. “The system’s defined and uniform mechanical stimulation is vastly different from how T cells are usually cultured—cells that attach to the bottom of a culture dish encounter a highly inelastic surface, while those remaining in suspension are surrounded by the viscous medium.”

Mechanical activity has natural effects

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 co-first author Kwasi Adu-Berchie, Ph.D., who completed his Ph.D. 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.”

The researchers discovered the activity of AP-1, a transcription factor that connects T cells’ receipt 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 increased, and genes that rely on them for expression were enriched, not only in T cells isolated from more elastic hydrogels, but also in T cells isolated from cancer and fibrotic tissues from patients, which are stiffer and more elastic than neighboring healthy tissues. They were able to counteract the effects of a more elastic collagen matrix on T cells by inhibiting one of AP-1’s components with a medication.

The scientists used therapeutic CAR-T cells engineered to bind a specific antigen of a human lymphoma cell line to study how diverse mechanical stimulations and T cells’ expected gene expression profiles translated into actual features and functions. CAR-T cells that were activated in vitro on a more elastic collagen matrix had a greater ability to destroy lymphoma cells. In addition, CAR-T cells activated in a more elastic matrix and adoptively transplanted into mice with the same kind of lymphoma were much more capable of lowering tumor burden and extending the animals’ lives than CAR-T cells produced in a less elastic matrix.

“This study merges three seemingly disparate fields, biomaterials, immunotherapy, and mechanobiology, to develop an entirely new form of biomaterials-based mechanotherapeutic. It is easy to see how these findings can potentially open up new avenues to improve adoptive T cell therapies for patients in the future,” said Wyss Founding Director Donald Ingber, M.D., Ph.D., who is also the Judah Folkman Professor of Vascular Biology at Harvard Medical School and Boston Children’s Hospital, and the Hansjörg Wyss Professor of Bioinspired Engineering at SEAS.

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