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Curiously, at the end of this complex selection process, CD4<jats:sup>+<\/jats:sup>cells invariably outnumber CD8<jats:sup>+<\/jats:sup>cells. Here, we examine the dynamics of repertoire formation and the emergence of the skewed CD4\/CD8 ratio using high-resolution endogenous CRISPR\/Cas9 barcoding that indelibly marks immature T cells at the DN2\/DN3 pre-TCR stage. In wild-type mice, greater clone size of CD4<jats:sup>+<\/jats:sup>cells and an intrinsically greater probability of Tcr \u03b2 clonotypes for pMHCII interactions are major contributors to the skewed CD4\/CD8 ratio. Clonal perturbations of thymocyte differentiation following the precocious expression of a rearranged iNKT invariant TCR \u03b1 chain are due to loss of Tcr \u03b2 clonotypes from the CD4 lineage-committed pre-selection repertoire. The present barcoding scheme offers a novel means to examine the clonal dynamics of lymphocyte differentiation orthogonal to that using TCR clonotypes.<\/jats:p>","DOI":"10.1038\/s42003-022-03870-3","type":"journal-article","created":{"date-parts":[[2022,9,5]],"date-time":"2022-09-05T18:02:33Z","timestamp":1662400953000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Clonal dynamics underlying the skewed CD4\/CD8 ratio of mouse thymocytes revealed by TCR-independent barcoding"],"prefix":"10.1038","volume":"5","author":[{"given":"Norimasa","family":"Iwanami","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7601-9873","authenticated-orcid":false,"given":"Malte","family":"Petersen","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Dagmar","family":"Diekhoff","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5172-7478","authenticated-orcid":false,"given":"Thomas","family":"Boehm","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"297","published-online":{"date-parts":[[2022,9,5]]},"reference":[{"key":"3870_CR1","doi-asserted-by":"crossref","first-page":"227","DOI":"10.1084\/jem.141.1.227","volume":"141","author":"H Shiku","year":"1975","unstructured":"Shiku, H. et al. Expression of T-cell differentiation antigens on effector cells in cell-mediated cytotoxicity in vitro. Evidence for functional heterogeneity related to the surface phenotype of T cells. J. Exp. Med. 141, 227\u2013241 (1975).","journal-title":"J. Exp. Med."},{"key":"3870_CR2","doi-asserted-by":"crossref","first-page":"1376","DOI":"10.1084\/jem.141.6.1376","volume":"141","author":"H Cantor","year":"1975","unstructured":"Cantor, H. & Boyse, E. A. Functional subclasses of T-lymphocytes bearing different Ly antigens. I. The generation of functionally distinct T-cell subclasses is a differentiative process independent of antigen. J. Exp. Med. 141, 1376\u20131389 (1975).","journal-title":"J. Exp. Med."},{"key":"3870_CR3","doi-asserted-by":"crossref","first-page":"219","DOI":"10.1038\/253219a0","volume":"253","author":"P Kisielow","year":"1975","unstructured":"Kisielow, P. et al. Ly antigens as markers for functionally distinct subpopulations of thymus-derived lymphocytes of the mouse. 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