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Immunol."],"abstract":"<jats:p>This study discusses substantive advances in T cell proliferation analysis, with the aim to provoke a re-evaluation of the generally-held view that Ki-67 is a reliable proliferation marker<jats:italic>per se<\/jats:italic>, and to offer a more sensitive and effective method for T cell cycle analysis, with informative examples in mouse and human settings. We summarize recent experimental work from our labs showing that, by Ki-67\/DNA dual staining and refined flow cytometric methods, we were able to identify T cells in the S-G<jats:sub>2<\/jats:sub>\/M phases of the cell-cycle in the peripheral blood (collectively termed \u201cT Double S\u201d for<jats:underline>T<\/jats:underline>cells in<jats:underline>S<\/jats:underline>-phase<jats:italic>in<jats:underline>S<\/jats:underline>anguine<\/jats:italic>: in short \u201cT<jats:sub>DS<\/jats:sub>\u201d cells). Without our refinement, such cells may be excluded from conventional lymphocyte analyses. Specifically, we analyzed clonal expansion of antigen-specific CD8 T cells in vaccinated mice, and demonstrated the potential of T<jats:sub>DS<\/jats:sub>cells to reflect immune dynamics in human blood samples from healthy donors, and patients with type 1 diabetes, infectious mononucleosis, and COVID-19. The Ki-67\/DNA dual staining, or T<jats:sub>DS<\/jats:sub>assay, provides a reliable approach by which human peripheral blood can be used to reflect the dynamics of human lymphocytes, rather than providing mere steady-state phenotypic snapshots. The method does not require highly sophisticated \u201c-omics\u201d capabilities, so it should be widely-applicable to health care in diverse settings. Furthermore, our results argue that the T<jats:sub>DS<\/jats:sub>assay can provide a window on immune dynamics in extra-lymphoid tissues, a long-sought potential of peripheral blood monitoring, for example in relation to organ-specific autoimmune diseases and infections, and cancer immunotherapy.<\/jats:p>","DOI":"10.3389\/fimmu.2021.653974","type":"journal-article","created":{"date-parts":[[2021,4,9]],"date-time":"2021-04-09T04:23:14Z","timestamp":1617942194000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":28,"title":["To Ki or Not to Ki: Re-Evaluating the Use and Potentials of Ki-67 for T Cell Analysis"],"prefix":"10.3389","volume":"12","author":[{"given":"Francesca","family":"Di Rosa","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Andrea","family":"Cossarizza","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Adrian 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