{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,4,11]],"date-time":"2026-04-11T20:44:13Z","timestamp":1775940253313,"version":"3.50.1"},"posted":{"date-parts":[[2025,3,11]]},"group-title":"Immunology","reference-count":78,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,3,11]],"date-time":"2025-03-11T00:00:00Z","timestamp":1741651200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,3,15]]},"abstract":"<jats:title>SUMMARY<\/jats:title>\n                <jats:p>\n                  Chimeric antigen receptor (CAR) T-cell therapy has expanded therapeutic options for patients with diffuse large B-cell lymphoma (DLBCL). However, progress in improving clinical outcomes has been limited by an incomplete understanding of CAR T-cell differentiation in patients. To comprehensively investigate CAR T-cell differentiation in vivo, we performed single-cell, multi-modal, and longitudinal analyses of CD28-costimulated CAR T cells from infusion product and peripheral blood (day 8-28) of patients with DLBCL who were successfully treated with axicabtagene ciloleucel. Here, we show that CD8\n                  <jats:sup>+<\/jats:sup>\n                  CAR T cells undergo two distinct waves of clonal expansion. The first wave is dominated by CAR T cells with an exhausted-like effector memory phenotype during the peak expansion period (day 8-14). The second wave is dominated by CAR T cells with a terminal effector phenotype during the post-peak persistence period (day 21-28). Importantly, the two waves have distinct ontogeny and are biologically uncoupled. Furthermore, lineage tracing analysis via each CAR T cell\u2019s endogenous TCR clonotype demonstrates that the two waves originate from different effector precursors in the infusion product. Precursors of the first wave exhibit more effector-like signatures, whereas precursors of the second wave exhibit more stem-like signatures. These findings suggest that pre-infusion heterogeneity mediates the two waves of in vivo clonal expansion. Our findings provide evidence against the intuitive idea that the post-peak contraction in CAR abundance is solely apoptosis or extravasation of short-lived CAR T cells from peak expansion. Rather, our findings demonstrate that CAR T-cell expansion and persistence are mediated by clonally, phenotypically, and ontogenically distinct CAR T-cell populations that serve complementary clinical purposes.\n                <\/jats:p>","DOI":"10.1101\/2025.03.05.641715","type":"posted-content","created":{"date-parts":[[2025,3,11]],"date-time":"2025-03-11T05:10:16Z","timestamp":1741669816000},"source":"Crossref","is-referenced-by-count":0,"title":["Two-Stage CD8\n                  <sup>+<\/sup>\n                  CAR T-Cell Differentiation in Patients with Large B-Cell Lymphoma"],"prefix":"10.64898","author":[{"given":"Guoshuai","family":"Cao","sequence":"first","affiliation":[{"name":"Pritzker School of Molecular Engineering, University of Chicago, Chicago, IL 60637, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yifei","family":"Hu","sequence":"additional","affiliation":[{"name":"Pritzker School of Medicine, University of Chicago, Chicago, IL 60637, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Tony","family":"Pan","sequence":"additional","affiliation":[{"name":"Pritzker School of Molecular Engineering, University of Chicago, Chicago, IL 60637, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Erting","family":"Tang","sequence":"additional","affiliation":[{"name":"Pritzker School of Molecular Engineering, University of Chicago, Chicago, IL 60637, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Nick","family":"Asby","sequence":"additional","affiliation":[{"name":"Pritzker School of Molecular Engineering, University of Chicago, Chicago, IL 60637, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Thomas","family":"Althaus","sequence":"additional","affiliation":[{"name":"The David and Etta Jonas Center for Cellular Therapy, University of Chicago, Chicago, IL 60637, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9286-6562","authenticated-orcid":false,"given":"Jun","family":"Wan","sequence":"additional","affiliation":[{"name":"Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN 46202, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Peter A.","family":"Riedell","sequence":"additional","affiliation":[{"name":"Committee on Cancer Biology, University of Chicago, Chicago, IL 60637, USA"},{"name":"Department of Medicine, University of Chicago, Chicago, IL 60637, USA"},{"name":"The David and Etta Jonas Center for Cellular Therapy, University of Chicago, Chicago, IL 60637, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Michael R.","family":"Bishop","sequence":"additional","affiliation":[{"name":"Department of Medicine, University of Chicago, Chicago, IL 60637, USA"},{"name":"The David and Etta Jonas Center for Cellular Therapy, University of Chicago, Chicago, IL 60637, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Justin P.","family":"Kline","sequence":"additional","affiliation":[{"name":"Committee on Immunology, University of Chicago, Chicago, IL 60637, USA"},{"name":"Committee on Cancer Biology, University of Chicago, Chicago, IL 60637, USA"},{"name":"Department of Medicine, University of Chicago, Chicago, IL 60637, USA"},{"name":"The David and Etta Jonas Center for Cellular Therapy, University of Chicago, Chicago, IL 60637, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jun","family":"Huang","sequence":"additional","affiliation":[{"name":"Pritzker School of Molecular Engineering, University of Chicago, Chicago, IL 60637, USA"},{"name":"Committee on Immunology, University of Chicago, Chicago, IL 60637, USA"},{"name":"Committee on Cancer Biology, University of Chicago, Chicago, IL 60637, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"54368","reference":[{"key":"2026041113015784000_2025.03.05.641715v2.1","unstructured":"Diffuse Large B-Cell Lymphoma - 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