High-dimensional spectral flow cytometry reveals distinct T cell profiles and thymopoiesis abnormalities in thymic epithelial lesions

Thymic epithelial lesions (TELs) are rare disorders that include thymic hyperplasia (TH), thymomas (Tys), and thymic carcinomas (TCs). Tys are further subdivided into histological subtypes A, AB, B1, B2, and B3, with a progressively worse outcome from type A to B3. TH and Tys are characterized by an active intralesional thymopoiesis and a strong association with autoimmune disorders (ADs). However, the precise mechanisms linking TELs and ADs remain poorly understood. To address this issue, we performed an in-depth characterization of intrathymic T cell development in 56 subjects (48 TEL patients and 8 pediatric healthy controls (HCs)), using a 39-color full spectrum flow cytometry panel specifically designed to investigate the multistep process on intrathymic T cell maturation. The panel, developed and optimized following best practices for high-dimensional flow cytometry, allows the simultaneous assessment of a huge number of key markers variably expressed during T cell development, thus enabling the identification of conventional and unconventional T cells, including γδ T and MAIT cell subsets, across distinct maturation stages. To analyze the complex high-dimensional data obtained with this panel, we employed a multi-step bioinformatic pipeline. A total of 7 millions of cells were first integrated and batch-corrected with pyHarmony, followed by a dimensionality reduction approach that allowed the visualization of T cell populations in reduced dimensions while preserving the structure of the data. The Leiden algorithm was then applied for unsupervised clustering to identify and characterize distinct T cell subsets across different TEL subtypes. Our results demonstrated active thymopoiesis in TH and type AB, B1, and B2 Tys, characterized by the presence of several immature thymocyte stages. Tys showed significant abnormalities in thymocyte maturation, including an increased frequency of CD4+ immature single-positive thymocytes and a significant reduction in regulatory T cells (Tregs), possibly involved in the autoimmune dysregulation commonly observed in these patients. Type AB Ty exhibited also additional features, including an enrichment of unconventional Vδ1 T cells and a disrupted balance between double-positive and single-positive thymocytes, with a reduced frequency of ready-to-egress CD4+ T cells. In contrast, type A and B3 Tys and TCs lacked active thymopoiesis, and were associated with a marked enrichment of Tregs and immune checkpoint-high MAIT cells in the tumor microenvironment, characterized by high expression of inhibitory molecules such as PD1, CD39, TIGIT and TIM3. These tumors were also enriched in tissue-resident memory CD8+ T cells, activated and terminally differentiated Vδ1 T cells, and CD161+ effector memory CD4+ T cells, thus indicating the concomitant presence of anti-inflammatory and pro-inflammatory immune populations in the tumor microenvironment that may have potential implications in the selection of specific checkpoint inhibition therapies. Overall, this study demonstrates profound differences in thymic T cell profiles among TEL subtypes that may help the comprehension of the immune dysregulation occurring in TEL patients and may pave the way for personalized therapeutic approaches in these rare diseases.