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To define the functional consequences of distinct STAT3\n                    <jats:sup>D661<\/jats:sup>\n                    variants, we integrated computational, structural and in\u00a0vitro and in\u00a0vivo genetic approaches. AlphaMissense and PolyPhen\u20102 classified all four STAT3\n                    <jats:sup>D661<\/jats:sup>\n                    variants (D661Y, D661V, D661N and D661H) as pathogenic. ClinVar classified D661Y and D661V as variants of uncertain significance. AlphaFold 3\u2010based modelling predicted that D661Y and D661V strongly promoted SH2\u2010TAD\u2010mediated dimerization, while D661N and D661H exerted weaker structural effects. Functional in\u00a0vitro assays in\n                    <jats:italic>Stat3<\/jats:italic>\n                    \u2010deficient T cells demonstrated a gain\u2010of\u2010function (GOF) hierarchy of the STAT3 variants (D661Y\u2009\u2248\u2009V\u2009&gt;\u2009H\u2009&gt;\u2009\n                    <jats:italic>N<\/jats:italic>\n                    ) resulting in activation of canonical STAT3 target genes and immune transcriptional programs. In\u00a0vivo, only STAT3\n                    <jats:sup>D661H<\/jats:sup>\n                    mice were viable, displaying reduced CD4\n                    <jats:sup>+<\/jats:sup>\n                    T cells, expansion of memory CD8\n                    <jats:sup>+<\/jats:sup>\n                    T cells and enhanced immune gene expression. Collectively, our findings define a gradient of STAT3 D661 GOF variants, consistent with in\u00a0vitro and in\u00a0vivo experiments. D661Y and D661V mutants exhibited stronger transcriptional activity in T cells with impaired viability of mice carrying these variants.\n                  <\/jats:p>","DOI":"10.1111\/jcmm.71015","type":"journal-article","created":{"date-parts":[[2026,1,13]],"date-time":"2026-01-13T08:37:57Z","timestamp":1768293477000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["STAT3 SH2 Domain Aspartic Acid 661 Mutations Activate Immune Gene Programs"],"prefix":"10.1111","volume":"30","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-7785-5942","authenticated-orcid":false,"given":"Hye Kyung","family":"Lee","sequence":"first","affiliation":[{"name":"Section of Genetics and Physiology, National Institute of Diabetes and Digestive and Kidney Diseases, US National Institutes of Health  Bethesda Maryland 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