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Here, we introduce a novel in vitro cultivation system based on ThymoSpheres (TS): dense spheroids consisting of DLL4\u2010expressing stromal cells and human hematopoietic precursor cells, in the absence of thymic epithelial cells. These spheroids are subsequently cultured at the air\u2013liquid interphase. TS generate large numbers of mature T cells, are easy to manipulate, scalable, and can be repeatably sampled to monitor T\u2010cell differentiation. The mature T cells generated from primary human hematopoietic precursor cells were extensively characterized using single\u2010cell RNA and combined T\u2010cell receptor (TCR) sequencing. These predominantly CD8\u03b1 T cells exhibit transcriptional and TCR CDR3 characteristics similar to the recently described human polyclonal \u03b1\u03b2 unconventional T cell (UTC) lineage. This includes the expression of hallmark genes associated with agonist selection, such as\n                    <jats:italic>IKZF2<\/jats:italic>\n                    (Helios), and the expression of various natural killer receptors. The TCR repertoire of these UTCs is polyclonal and enriched for CDR3\u2010associated autoreactive features and early rearrangements of the TCR\u2010\u03b1 chain. In conclusion, TS cultures offer an intriguing platform to study the development of this human polyclonal UTC lineage and its inducing selection mechanisms.\n                  <\/jats:p>","DOI":"10.1002\/eji.202451265","type":"journal-article","created":{"date-parts":[[2024,9,9]],"date-time":"2024-09-09T04:08:40Z","timestamp":1725854920000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["ThymoSpheres culture: A model to study human polyclonal unconventional T cells"],"prefix":"10.1002","volume":"54","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-9372-1920","authenticated-orcid":false,"given":"Lore","family":"Billiet","sequence":"first","affiliation":[{"name":"Department of Diagnostic Sciences Ghent University  Ghent Belgium"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Hanne","family":"Jansen","sequence":"additional","affiliation":[{"name":"Department of Diagnostic Sciences Ghent University  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