ThymoSpheres culture: a model to study human polyclonal unconventional T cells

In vitro cultures remain crucial for studying the fundamental mechanisms of human T cell development. Here, we introduce a novel in vitro cultivation system based on ThymoSpheres (TS): dense spheroids of DLL4 expressing stromal cells and human hematopoietic precursor cells (HPCs), in the absence of thymic epithelial cells. These spheroids are subsequently cultured at the air-liquid interphase. TS generate large numbers of mature T cells, are easy to manipulate, scalable, and can be repeatably sampled to monitor T cell differentiation. The mature T cells generated from primary human HPCs were extensively characterized using single-cell RNA and combinedT cell receptor (TCR) sequencing. These predominantly CD8α T cells exhibit transcriptional and CDR3 characteristics recently described in a human polyclonal αβ unconventional T cell (UTC) lineage. This includes the expression of hallmark genes associated with agonist selection, such as IKZF2(Helios), and expression of various Natural Killer (NK) receptors. The TCR repertoire of these UTCs is polyclonal and enriched for CDR3-associated autoreactive features and early rearrangements of the TCRα chain. In conclusion, TS cultures offer an intriguing platform to study the development of this human polyclonal UTC lineage and its inducing selection mechanisms. Multiple TS-containing inserts were harvested and pooled after 9 weeks of culture. The CD3+ TCRγδ- fraction was sorted for single-cell analysis.