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Confirming that complemented organs and cells work normally in chimeric animals is critical to demonstrating the feasibility of blastocyst complementation. Here, we generated thymus-complemented chimeric mice, assessed the efficacy of anti-PD-L1 antibody in tumor-bearing chimeric mice, and then investigated T-cell function. Thymus-complemented chimeric mice were generated by injecting C57BL\/6 (B6) embryonic stem cells into <jats:italic>Foxn1<\/jats:italic><jats:sup><jats:italic>nu<\/jats:italic>\/<jats:italic>nu<\/jats:italic><\/jats:sup> morulae or blastocysts. Flow cytometry data showed that the chimeric mouse thymic epithelial cells (TECs) were derived from the B6 cells. T cells appeared outside the thymi. Single-cell RNA-sequencing analysis revealed that the TEC gene-expression profile was comparable to that in B6 mice. Splenic T cells of chimeric mice responded very well to anti-CD3 stimulation in vitro; CD4<jats:sup>+<\/jats:sup> and CD8<jats:sup>+<\/jats:sup> T cells proliferated and produced IFN\u03b3, IL-2, and granzyme B, as in B6 mice. Anti-PD-L1 antibody treatment inhibited MC38 tumor growth in chimeric mice. Moreover, in the chimeras, anti-PD-L1 antibody restored T-cell activation by significantly decreasing PD-1 expression on T cells and increasing IFN\u03b3-producing T cells in the draining lymph nodes and tumors. T cells produced by complemented thymi thus functioned normally in vitro and in vivo. To successfully generate humanized animals by blastocyst complementation, both verification of the function and gene expression profiling of complemented organs\/cells in interspecific chimeras will be important in the near future.<\/jats:p>","DOI":"10.1038\/s41598-022-07159-7","type":"journal-article","created":{"date-parts":[[2022,2,25]],"date-time":"2022-02-25T11:10:45Z","timestamp":1645787445000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":9,"title":["In vitro and in vivo functions of T cells produced in complemented thymi of chimeric mice generated by blastocyst complementation"],"prefix":"10.1038","volume":"12","author":[{"given":"Kazuto","family":"Yamazaki","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kenji","family":"Kubara","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Satoko","family":"Ishii","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Peng","family":"Li","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ryo","family":"Dairiki","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Taro","family":"Hihara","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuta","family":"Ishizuka","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yukina","family":"Izumi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Minoru","family":"Kumai","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tsutomu","family":"Kamisako","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hiroyoshi","family":"Ishizaki","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hideyuki","family":"Sato","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hideki","family":"Masaki","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Naoaki","family":"Mizuno","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kaoru","family":"Mitsuhashi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Masashi","family":"Ito","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sanae","family":"Hamanaka","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tomoyuki","family":"Yamaguchi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Motoo","family":"Watanabe","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fumihiro","family":"Sugiyama","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hiromitsu","family":"Nakauchi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2022,2,25]]},"reference":[{"key":"7159_CR1","doi-asserted-by":"publisher","first-page":"4528","DOI":"10.1073\/pnas.90.10.4528","volume":"90","author":"J Chen","year":"1993","unstructured":"Chen, J., Lansford, R., Stewart, V., Young, F. & Alt, F. 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