{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,4,11]],"date-time":"2026-04-11T07:57:36Z","timestamp":1775894256741,"version":"3.50.1"},"posted":{"date-parts":[[2025,3,13]]},"group-title":"Immunology","reference-count":62,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,3,13]],"date-time":"2025-03-13T00:00:00Z","timestamp":1741824000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,3,13]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  CD28 ligation provides critical signals that modulate activated T cell fate. In a human to mouse reverse-engineering approach, a single amino acid substitution adjacent to the C-terminal proline-rich domain created CD28\n                  <jats:sup>A210P<\/jats:sup>\n                  mice with enhanced signaling. CD28\n                  <jats:sup>A210P<\/jats:sup>\n                  mice experienced pro-inflammatory responses to CD28 superagonist antibody, analogous to severe cytokine storm induced in a human clinical trial, with a striking increase of activated CD8 T cells. In acute and chronic viral infections, early activation and expansion of CD28\n                  <jats:sup>A210P<\/jats:sup>\n                  CD8 effector T cells increased, with accelerated exhaustion in chronic infection. Mechanistically, CD28\n                  <jats:sup>A210P<\/jats:sup>\n                  enhanced JunB, IL-2, and inhibitory receptors driven by MEK1\/2. Generation of CD28\n                  <jats:sup>A210P<\/jats:sup>\n                  stem-like progenitor (Tpex) cells was enhanced in acute and chronic infections, and further expanded by PD-L1 blockade in chronically-infected mice. Thus, \u2018humanized\u2019 PYAP mice reveal key roles for CD28 signaling strength in CD8 activation, accelerating exhaustion during antigen persistence, while promoting and sustaining Tpex during acute and chronic viral infection.\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>One sentence Summary<\/jats:title>\n                  <jats:p>A single amino acid substitution adjacent to PYAP to \u2018humanize\u2019 CD28 signaling enhances superagonist response, early CD8 activation and Tpex generation during viral infection while accelerating exhaustion and sustaining Tpex during chronic infection.<\/jats:p>\n                  <jats:fig id=\"ufig1\" position=\"float\" orientation=\"portrait\" fig-type=\"figure\">\n                    <jats:label>Graphical Abstract:<\/jats:label>\n                    <jats:caption>\n                      <jats:title>\u2018Humanized\u2019 CD28 PYAPP enhances numbers of CD8 T cell effectors and stem-like precursors during acute viral infection, and accelerates exhaustion while sustaining increased self-renewing Tpex cells that are favored during PD-L1 blockade.<\/jats:title>\n                    <\/jats:caption>\n                    <jats:graphic xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" xlink:href=\"642460v1_ufig1\" position=\"float\" orientation=\"portrait\"\/>\n                  <\/jats:fig>\n                <\/jats:sec>","DOI":"10.1101\/2025.03.10.642460","type":"posted-content","created":{"date-parts":[[2025,3,13]],"date-time":"2025-03-13T23:30:36Z","timestamp":1741908636000},"source":"Crossref","is-referenced-by-count":0,"title":["Humanizing a CD28 signaling domain affects CD8 activation, exhaustion and stem-like precursors"],"prefix":"10.64898","author":[{"given":"Alexander E.","family":"Brady","sequence":"first","affiliation":[{"name":"Cornell University, Ithaca NY, 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