The thymic microenvironment is comprised of a range of thymic stromal cells and is a crucial requirement for T cell development. As thymocytes migrate through different microenvironments of the thymus, they receive the necessary signals for development. Although there are various populations of thymic stromal cells, the range of cell types in this compartment is not yet fully characterized. Moreover, much about their exact functions and how they guide T cell development remains poorly understood.
We hypothesised that unbiased profiling of cell types that make up the thymic stroma and how they physically interact with developing thymocytes will allow a better understanding of their roles in T cell development. To achieve this, we exploited the phenomenon of thymic rosettes, which are in vivo cellular complexes composed of 10-20 developing thymocytes surrounding with a central stromal cell. They represent a snapshot of the physical interactions between thymocytes and stroma and can be isolated intact from the thymus.
To characterize the composition of rosettes, we employed flow cytometry analysis and single cell RNA sequencing on the 10X Genomics platform. Thymocytes at post-positive selection stages of development were enriched in rosettes, suggesting that interactions with stromal cells in rosettes are particularly important in the later stages. Furthermore, single cell RNA sequencing revealed previously unknown complexities in the thymic B cells, dendritic cells and macrophages. They displayed gene signatures unique to the thymus, suggesting that they have specialised and unique functions for guiding T cell development. The thymic B cells in rosettes were distinct from splenic B cell subsets and were capable of differentiating into plasma cells. Monocyte and macrophage populations in rosettes represented a linear differentiation process, and the intermediate stages appeared to be dendritic cell-like. There appeared to be four functionally distinct subsets of thymic dendritic cells, instead of three as previously identified, and these subsets displayed subset-specific chemokine receptor expression.
These new insights into thymic rosettes help to establish a platform for mapping specific interactions in individual rosettes using high throughput sequencing techniques. Ultimately, delineating these interactions helps to complete understanding of T cell development.