{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,8,4]],"date-time":"2026-08-04T13:55:21Z","timestamp":1785851721766,"version":"3.56.0"},"posted":{"date-parts":[[2026,8,1]]},"group-title":"Immunology","reference-count":71,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,8,1]],"date-time":"2026-08-01T00:00:00Z","timestamp":1785542400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,8,1]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  Immune checkpoint inhibitors (ICI) that are antibodies against CTLA-4 have achieved therapeutic effects on multiple types of cancers\n                  <jats:sup>1\u20134<\/jats:sup>\n                  , but the mechanisms underlying the therapy remain incompletely understood. In contrast to the initial theory that the antibody blockades CTLA-4 on T cells is of central importance, it has recently been demonstrated that selective reduction of CD4\n                  <jats:sup>+<\/jats:sup>\n                  Foxp3\n                  <jats:sup>+<\/jats:sup>\n                  regulatory T cells (Tregs) in the tumor microenvironment by the antibody plays a key role in the anti-tumor effects\n                  <jats:sup>5\u20138<\/jats:sup>\n                  , although this phenomenon remains under debate in human patients\n                  <jats:sup>9\u201312<\/jats:sup>\n                  . We show here that anti-CTLA-4 antibody engages CTLA-4 in Tregs specifically in the tumor tissues to reduce their stability and survival and weaken their suppressive function through upregulating TGF-\u03b2 signaling. This leads to the antibody-mediated - upregulation of CD4\n                  <jats:sup>+<\/jats:sup>\n                  and CD8\n                  <jats:sup>+<\/jats:sup>\n                  effector T cell anti-tumor immunity and consequently cancer immunotherapy. Specifically, anti-CTLA-4 antibody directly stimulates CTLA-4 in intratumor Tregs to enhance their TGF-\u03b2 receptor I (T\u03b2RI)-mediated TGF-\u03b2 signaling\n                  <jats:sup>13<\/jats:sup>\n                  . This results in reduction of IL-2 receptor CD25 expression and downregulation of lactate metabolism in intratumor Tregs to reduce their stability and survival\n                  <jats:sup>14\u201316<\/jats:sup>\n                  , and also compromises their suppressive function by inhibiting Foxp3 expression. This activity requires high levels of CTLA-4 expression in the intratumor Tregs and the presence of antibody Fc receptors in the tumor tissues. Strikingly, deletion of T\u03b2RI specifically in Tregs completely prevents the reduction of intratumor Tregs and abrogates the anti-CTLA-4-mediated cancer immunotherapy. In contrast to intratumor Tregs, anti-CTLA-4 antibody treatment blocks CTLA-4 in the intratumor CD4\n                  <jats:sup>+<\/jats:sup>\n                  Foxp3\n                  <jats:sup>\u2212<\/jats:sup>\n                  and CD8\n                  <jats:sup>+<\/jats:sup>\n                  T effector cells and in the peripheral Tregs to decrease their T\u03b2RI expression and increase their expansion due to their relatively low levels of CTLA-4 in the same tumor bearing mice. Significantly, the engagement of CTLA-4 by anti-human CTLA-4 antibody (ipilimumab) also upregulates\n                  <jats:italic>TGFBR1<\/jats:italic>\n                  but decreases\n                  <jats:italic>Il2RA<\/jats:italic>\n                  expression and lactate metabolism in human Tregs\n                  <jats:italic>in vitro<\/jats:italic>\n                  and in humanized mice\n                  <jats:italic>in vivo<\/jats:italic>\n                  , leading to suppression of tumor progression. We have provided additional mechanism underlying anti-CTLA-4-mediated anti-tumor effects through destabilizing intratumor Tregs by CTLA-4 engagement-mediated TGF-\u03b2 signaling. This could lay a theoretical foundation for designing optimal immunotherapy based on anti-CTLA-4 antibody in cancer patients.\n                <\/jats:p>","DOI":"10.64898\/2026.07.28.741328","type":"posted-content","created":{"date-parts":[[2026,8,2]],"date-time":"2026-08-02T17:05:24Z","timestamp":1785690324000},"source":"Crossref","is-referenced-by-count":0,"title":["Destabilization of intratumor Tregs by CTLA-4 engagement confers anti-CTLA-4-driven immunotherapy"],"prefix":"10.64898","author":[{"given":"Na","family":"Liu","sequence":"first","affiliation":[{"name":"Mucosal Immunology Section, National Institute of Dental and Craniofacial Research, National Institute of health, Bethesda, MD, 20892, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Junji","family":"Xu","sequence":"additional","affiliation":[{"name":"Mucosal Immunology Section, National Institute of Dental and Craniofacial Research, National Institute of health, Bethesda, MD, 20892, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8859-6771","authenticated-orcid":false,"given":"Wai Lim","family":"Ku","sequence":"additional","affiliation":[{"name":"Laboratory of Epigenome Biology, Systems Biology Center, National Heart, Lung and Blood Institute, NIH, Bethesda, MD, 20892, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Weiwei","family":"Chen","sequence":"additional","affiliation":[{"name":"Mucosal Immunology Section, National Institute of Dental and Craniofacial Research, National Institute of health, Bethesda, MD, 20892, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4665-1517","authenticated-orcid":false,"given":"Yaqiang","family":"Cao","sequence":"additional","affiliation":[{"name":"Laboratory of Epigenome Biology, Systems Biology Center, National Heart, Lung and Blood Institute, NIH, Bethesda, MD, 20892, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rida","family":"Kazmi","sequence":"additional","affiliation":[{"name":"Mucosal Immunology Section, National Institute of Dental and Craniofacial Research, National Institute of health, Bethesda, MD, 20892, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wenwen","family":"Jin","sequence":"additional","affiliation":[{"name":"Mucosal Immunology Section, National Institute of Dental and Craniofacial Research, National Institute of health, Bethesda, MD, 20892, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Thierry","family":"Gauthier","sequence":"additional","affiliation":[{"name":"Mucosal Immunology Section, National Institute of Dental and Craniofacial Research, National Institute of health, Bethesda, MD, 20892, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shunqun","family":"Luo","sequence":"additional","affiliation":[{"name":"Center for Vascular and Inflammatory Diseases, Department of Surgery, University of Maryland School of Medicine, Baltimore, MD 21201, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Song","family":"Shen","sequence":"additional","affiliation":[{"name":"Mucosal Immunology Section, National Institute of Dental and Craniofacial Research, National Institute of health, Bethesda, MD, 20892, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Liliana C.","family":"Pati\u00f1o","sequence":"additional","affiliation":[{"name":"Mucosal Immunology Section, National Institute of Dental and Craniofacial Research, National Institute of health, Bethesda, MD, 20892, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yun-Ji","family":"Lim","sequence":"additional","affiliation":[{"name":"Mucosal Immunology Section, National Institute of Dental and Craniofacial Research, National Institute of health, Bethesda, MD, 20892, 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