DISSECTING IMMUNE DYSREGULATION IN PATIENTS WITHTHYMIC EPITHELIAL TUMORS BY AUTOANTIBODY ANDSINGLE-CELL PROFILING

Thymic epithelial lesions (TELs) are a heterogeneous and rare group of diseases arising from the epithelial cells of the thymus. TELs include both benign conditions, such as thymic hyperplasia (TH), and malignant lesions, collectively referred to as thymic epithelial tumors (TETs). According to the 2021 WHO classification, TETs are further subdivided into thymomas comprising type A, AB, B1, B2, and B3 and thymic carcinomas (TC), based on tumor architecture, the morphology of malignant cells and the proportion of intratumoral immune cells. Prognosis progressively worsened from type A thymomas to TCs. Patients with thymomas showed an elevated susceptibility to paraneoplastic syndromes, in particular autoimmune diseases (ADs), which affects around 30% of thymoma patients. Evidence indicates that thymomas, but not TCs, can sustain intratumoral T-cell development, a process that appears dysregulated and likely contributes to thymoma-associated immune dysfunction, although the precise mechanisms linking TETs to ADs remain unclear. To address these knowledge gaps, we combined complementary approaches, including clinical and high-throughput autoAbs screening, a 39-color spectral flow cytometry panel to deeply characterize αβ, γδ and MAIT compartments, and single-cell RNA sequencing paired with αβ TCR sequencing to reconstruct developmental trajectories, functional states and clonal relationships. Pediatric thymuses were used as the healthy tissue counterpart whereas PBMCs from age-matched healthy donors were used as healthy peripheral blood controls. Public single-cell datasets were integrated to enable direct transcriptomic comparisons between patients and healthy individuals. Our results indicate that TEL patients can be classified into two main categories. One group, comprising TH and type AB, B1, B2 thymomas, retained intratumoral thymopoiesis, whereas type A, A-MNT, B3 thymomas and TC were predominantly infiltrated by mature T cells. Within this spectrum each histotype showed distinctive features: B1 thymomas supported efficient thymopoiesis and released CD4, CD8 and Vδ1 recent thymic emigrants (RTE) cells into the blood stream; B2 thymomas mirrored the intratumoral program but released predominantly Vδ1 RTE cells, indicating lower output of conventional naïve T cells; AB displayed a developmental bottleneck with accumulation of immature thymocytes and reduced naïve CD4 and CD8 pools; B3 thymomas showed little αβ thymopoiesis by flow cytometry but residual activity detectable by single-cell analysis; A thymomas and TCs resembled typical solid tumors dominated by memory T cells, with TCs uniquely enriched in highly immunosuppressive Tregs and exhausted TRM-like CD8 cells. Serology revealed that immune dysregulation extends beyond overt disease. Clinical screening detected autoAbs in most patients without symptoms and anti-AChR was frequent in asymptomatic thymoma, indicating tolerance breach before clinical onset. A high-content autoantigen array uncovered a distinct IgG cluster almost entirely composed of AB and B thymomas without overt ADs, characterized by anti-dsDNA, anti-IL-12, anti-AQP4 and anti-TIF1γ autoAbs with strong internal correlations. In contrast, IgM autoreactivity was reduced in AB and B thymomas yet increased in TH, consistent with sustained unswitched memory B cells and long-term IgM secretion. Together these findings support a model in which aberrant intratumoral thymopoiesis fails to enforce central tolerance, seeds autoreactive T cells and promotes autoreactive B-cell activation. Regulatory T cells (Treg) provided an additional link to tolerance failure. Within tumors we identified a developing and mature Treg subsets, both depleted in AB and B1 thymomas. Circulating Tregs were also reduced, supporting the idea of an impaired Treg generation and maintenance. This aligns with a possible defective peripheral B-cell tolerance checkpoints and offers a plausible route from local thymic dysfunction to systemic autoantibody production. TCs illustrated a different but targetable immune state, in which TRM-like cells retained cytotoxic programs but co-expressed multiple inhibitory receptors such as PD1, TIM3, CD38 and CD39, supporting the investigation of PD1 and TIM3 blockade as strategies to reinvigorate resident effectors. Paired scTCR analyses revealed clonal bridges between tumor and blood in all groups. In AB, B1 and B2 thymoma patients, shared clones were often singletons in tumor while expanded in blood, consistent with new emigrants arising intratumorally and expanding systemically. Recurrent clonotypes across individuals suggest convergent antigenic selection. Taken together, our integrated serological, spectral flow cytometric, and scRNAseq analyses provide a comprehensive immunological characterization of TEL patients linking disordered intratumoral T-cell development, failures of Treg and B-cell tolerance checkpoints and checkpoint-restrained resident effector programs to the dual clinical phenotype of autoimmunity and cancer. Future work should define the epithelial cues that misroute thymopoiesis, map the antigens driving shared clonotypes and test mechanism-guided interventions in prospective cohorts.