{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,6,23]],"date-time":"2026-06-23T16:58:15Z","timestamp":1782233895492,"version":"3.54.5"},"posted":{"date-parts":[[2026,2,5]]},"group-title":"Bioengineering","reference-count":48,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,2,5]],"date-time":"2026-02-05T00:00:00Z","timestamp":1770249600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/publicdomain\/zero\/1.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,6,21]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  Chimeric antigen receptor (CAR) T-cell efficacy depends critically on the costimulatory domain, which shapes downstream signaling and the immunophenotype of manufactured products. Despite mechanistic evidence that immune receptors function as motif-based signaling scaffolds, CAR engineering has focused on a narrow set of costimulatory domains\u2014principally CD28 and 4-1BB\u2014leaving much of the available signaling design space unexplored. Here, we screened 1,243 naturally occurring intracellular domains as costimulatory modules in an anti-CD20 CAR backbone in primary human CD8\n                  <jats:sup>+<\/jats:sup>\n                  T cells and quantified construct enrichment across memory-differentiation and PD-1-defined immunophenotypic compartments. Using Eukaryotic Linear Motif (ELM) annotations, we analyzed motif-phenotype associations via complementary statistical approaches: Mann\u2013Whitney screening and negative binomial regression identified ELM features associated with differential construct representation, while Dirichlet-Multinomial modeling\u2014which properly accounts for the compositional structure of FACS-partitioned data\u2014revealed that individual ELMs do not significantly alter phenotype distributions. This discrepancy indicates that single motifs primarily affect proliferation or survival rather than differentiation fate. In contrast, construct-level analysis using a leave-one-out compositional test identified specific costimulatory domains with significant phenotype-shifting effects, demonstrating that particular\n                  <jats:italic>combinations<\/jats:italic>\n                  of ELMs\u2014rather than individual motifs\u2014determine immunophenotype. These results suggest that CAR T-cell differentiation state is governed by the integrated output of multiple signaling motifs and provide a combinatorial framework for rational costimulatory domain engineering.\n                <\/jats:p>","DOI":"10.64898\/2026.02.01.700582","type":"posted-content","created":{"date-parts":[[2026,2,5]],"date-time":"2026-02-05T19:30:33Z","timestamp":1770319833000},"source":"Crossref","is-referenced-by-count":0,"title":["A motif-vocabulary model of CAR T-cell intracellular domains identifies determinants of immunophenotype differentiation"],"prefix":"10.64898","author":[{"given":"Conor","family":"Kelly","sequence":"first","affiliation":[{"name":"School of Medicine, Georgetown University, Washington, DC 20007"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Reem","family":"Bahr","sequence":"additional","affiliation":[{"name":"School of Medicine, Georgetown University, Washington, DC 20007"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wenjun","family":"Zhu","sequence":"additional","affiliation":[{"name":"Translational Stem Cell Biology Branch, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, MD"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Keyvan","family":"Keyvanfar","sequence":"additional","affiliation":[{"name":"Flow Cytometry Core, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, MD"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Praeep","family":"Dagur","sequence":"additional","affiliation":[{"name":"Flow Cytometry Core, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, MD"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Stefan","family":"Cordes","sequence":"additional","affiliation":[{"name":"Translational Stem Cell Biology Branch, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, MD"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026062309351431000_2026.02.01.700582v2.1","doi-asserted-by":"publisher","DOI":"10.1056\/NEJMoa1407222"},{"key":"2026062309351431000_2026.02.01.700582v2.2","doi-asserted-by":"publisher","DOI":"10.1056\/NEJMoa1707447"},{"key":"2026062309351431000_2026.02.01.700582v2.3","doi-asserted-by":"publisher","DOI":"10.1056\/NEJMoa2024850"},{"key":"2026062309351431000_2026.02.01.700582v2.4","doi-asserted-by":"publisher","DOI":"10.1038\/s41591-018-0010-1"},{"key":"2026062309351431000_2026.02.01.700582v2.5","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-021-04390-6"},{"key":"2026062309351431000_2026.02.01.700582v2.6","doi-asserted-by":"publisher","DOI":"10.1038\/s41591-022-02017-5"},{"key":"2026062309351431000_2026.02.01.700582v2.7","doi-asserted-by":"publisher","DOI":"10.1038\/s41573-019-0041-4"},{"key":"2026062309351431000_2026.02.01.700582v2.8","unstructured":"Mueller, K. 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