Graves\u27 orbitopathy (GO), the most severe manifestation of Graves\u27 hyperthyroidism (GH), is an autoimmune-mediated inflammatory disorder, and treatments often exhibit a low efficacy. CD4+ T cells have been reported to play vital roles in GO progression. To explore the pathogenic CD4+ T cell types that drive GO progression, we applied single-cell RNA sequencing (scRNA-Seq), T cell receptor sequencing (TCR-Seq), flow cytometry, immunofluorescence and mixed lymphocyte reaction (MLR) assays to evaluate CD4+ T cells from GO and GH patients. scRNA-Seq revealed the novel GO-specific cell type CD4+ cytotoxic T lymphocytes (CTLs), which are characterized by chemotactic and inflammatory features. The clonal expansion of this CD4+ CTL population, as demonstrated by TCR-Seq, along with their strong cytotoxic response to autoantigens, localization in orbital sites, and potential relationship with disease relapse provide strong evidence for the pathogenic roles of GZMB and IFN-γ-secreting CD4+ CTLs in GO. Therefore, cytotoxic pathways may become potential therapeutic targets for GO
