A high-throughput-compatible animal-cell-free miniaturised thymic organoid model for thymus biology studies and in vitro T cell production.

T cells, a type of white blood cell, are an essential component of our immune system. They coordinate and effect our immune responses so we can control infections. Controlling different infections requires different types of immune response and our T cells allow us to make specific immune responses to specific infections. Recently, the power of T cells has been harnessed in medicine to make a new type of therapy called immunotherapy. Immunotherapies are still in their infancy, but have already been used successfully to treat some blood cancers.

Within the body, T cells can only be made in a highly specialised organ, the thymus. The thymus instructs precursor cells in the blood to become T cells, then guides the developing T cells through a series of screening processes that ensure that only safe, functional T cells leave the thymus to become part of the immune system. These processes are needed because each T cell has on its surface a protein called T cell receptor (TCR). The TCR recognises and binds a small part of a specific protein (called a peptide) on the surface of other, non-T, cells. When T cells are developing, a very large number of different TCRs are made, that each recognise a peptide from a different protein. Each T cell has a different TCR. Some of the TCRs that are made can bind peptides from proteins from infectious agents such as viruses. Other TCRs can bind peptides from proteins in our own bodies. If these 'self-reactive' TCRs became part of our immune systems they would cause autoimmunity and to avoid this, they are screened out in the thymus. This screening is performed by special cells in the thymus called thymic epithelial cells, which can selectively remove or disarm 'self-reactive' T cells.

The thymus is one of the first organs to degenerate in healthy individuals, and this contributes to a general decline in immune system function with age. This is one of the major reasons that as we get older we become more susceptible to new infections, such as flu and covid19. Thymus degeneration also causes problems for adult patients requiring a bone marrow transplant (BMT). This is because, after transplant, some patients take several years to make enough T cells again and these individuals remain vulnerable to infections until their immune system has been properly rebuilt. If their thymus function could be boosted, this time would be shortened.

All of this together means there is a lot of interest in making T cells in the lab (eg. for immunotherapy) and in developing methods for boosting thymus function in patients (including BMT patients and the elderly), to increase their ability to fight new infections. However, at present, the only experimental models that mimic thymus function sufficiently well for these purposes rely on the use of cells obtained from the native thymus. Thymus tissue is scarce and hard to work with, which severely limits the numbers and size of studies that can currently be performed - for instance high throughput screening for new drugs relevant to thymus regeneration is not currently possible.
We have recently shown that thymic epithelial cells can be made in the lab, starting from stem cells. We have also shown that we can grow miniaturized thymus organs (MTOs), in the lab, starting from native thymus tissue. We make these MTOs in a format that is suitable for low, medium and high throughput studies including drug screening and lab based T cell production. However, their use is still limited by tissue supply. This project will test whether we can combine these two approaches to make fully animal-cell-free MTOs from stem cells. If successful, this new animal-cell-free model system will significantly reduce the number of animals used in research. It will also enable a new era of thymus research in which much larger scale experiments can easily be performed, opening up this area to a wide range of laboratories including the pharmaceutical industry.

Our ability to produce clinically useful T cell repertoires and to study T cell development and thymus biology in vitro is limited by the lack of a scalable physiological in vitro model amenable to genetic modification and to medium and high throughput use. All current physiologically-relevant in vitro models rely on ex vivo animal tissue and are necessarily low throughput due to tissue scarcity. Development of a validated, scalable animal-cell-free model that supports the in vitro production of physiologically-selected T cell repertoires and enables the study of physiological thymus biology would be transformative for the field, and would dramatically reduce or eliminate the use of animals in T cell and thymus-related research and translational studies.

We have shown that thymic epithelial cells (TEC), the principal thymic component required to mediate T cell repertoire development, can be generated in vitro by direct reprogramming of fibroblasts or pluripotent stem cells into induced TEC (iTEC) (Blackburn, unpublished). We have also recently developed a microwell-based physiological, miniaturised thymic organoid (MTO) model, based on ex vivo fetal TEC, that can support T cell development in vitro (Blackburn lab, unpublished).

We now propose to further develop MTO into an animal-cell-free model that is highly scalable and is also amenable to genetic modification. This novel model will be based on two pluripotent stem cell (PSC)-derived cell populations - PSC-derived iTEC and PSC-derived haematopoietic progenitor cells - rather than ex vivo cells. Once achieved, it will provide for the first time a physiologically-relevant, animal-cell-free model suitable for studying thymus biology and T cell development in vitro and amenable to low, medium and high-throughput study designs. This will result in a significant reduction in animal use in such studies, since current methodologies rely wholly or partly on the use of ex vivo cells.