Investigation of tertiary lymphoid structures to address autoimmunity in thymic epithelial tumors

Thymic epithelial tumors (TETs) are rare and heterogeneous tumors that arise from thymic epithelial cells (TECs). They include thymomas, further classified into 5 different subtypes (A, AB, B1, B2 and B3), and thymic carcinoma. Thymomas are characterized by an active intratumor thymopoiesis and by a strong association with autoimmune disorders (ADs), with myasthenia gravis affecting approximately 30% of patients. Notably, ADs are more frequently observed in patients with type B thymomas, further suggesting a potential link between specific histological subtypes and immune dysregulation. However, the precise pathophysiological mechanisms underlying the association between TETs and ADs, and the role of B cells in this context, are currently poorly understood. To investigate the involvement of B cells in TETs we first performed histopathological and immunohistochemical analyses on 126 TET cases. Hematoxylin and eosin (H&E) staining revealed the presence of lymphoid aggregates with features consistent with tertiary lymphoid structures (TLSs) in a significant proportion of cases, particularly among type B thymomas. The presence of these structures was further confirmed by staining these sections with the B cell marker CD20, which demonstrated dense B cell-rich areas suggestive of follicle-like organization. Overall, TLS-like structures were identified in 83% of type B thymomas. To further characterize these lymphoid structures at single-cell resolution, we analyzed the tumor microenvironment of 19 TETs using single-cell RNA sequencing. Among several immune populations, we annotated three main B cell clusters based on canonical genes such as MS4A1, CD19, XBP1, and SDC1. Among the different clusters, one exhibited a transcriptional profile resembling pre-pro B cells, expressing early developmental genes including RAG1, RAG2, DNTT, and VPREB1, suggesting the presence of developing B cells within the tumor. Another B cell cluster displayed a germinal center-like phenotype, characterized by the expression of BCL6, AICDA, and RGS13, indicative of local B cell activation and maturation. These populations were particularly enriched in B2 and B3 thymomas, providing molecular evidence for the presence of organized TLSs within the tumor microenvironment. These preliminary findings support a potential link between B cell organization in thymomas and autoimmune manifestations. The presence of highly organized lymphoid structures and both immature and germinal center–like B cell populations within TETs suggests ongoing intratumoral B cell maturation and activation processes. To further dissect the spatial architecture and cellular interactions within TLS-like structures, we plan to perform spatial transcriptomic analyses. This approach may provide novel insights into the comprehension of the immunopathogenic mechanisms underlying thymoma-associated autoimmunity. Thymic epithelial tumors (TETs) are rare and heterogeneous tumors that include thymic carcinoma and five thymoma subtypes (A, AB, B1, B2, B3). Thymomas are characterized by an active intratumor thymopoiesis and by a strong association with autoimmune disorders (AD). Notably, ADs are more frequent in patients with type B thymomas, further suggesting a potential link between specific hihistotypes and immune dysregulation. However, the precise pathophysiological mechanisms underlying this association and the involvment of B-cells, are currently poorly understood. To investigate B-cell involvement we performed histopathological and immunohistochemical analyses on 126 TET cases. Hematoxylin and eosin staining revealed the presence of tertiary lymphoid structures (TLSs) in a significant proportion of cases, particularly among type B thymomas. The presence of these structures was further confirmed by staining these sections with the B-cell marker CD20. Overall, TLS-like structures were identified in 83% of type B thymomas. To characterize these lymphoid structures at single-cell resolution, we analyzed the tumor microenvironment of 19 TETs using single-cell-RNA-sequencing. Unsupervised clustering identified three principal B-cell populations, defined by canonical B-cell genes MS4A1, CD19 and SDC1. Among the different clusters, one exhibited a transcriptional profile resembling pre-pro B-cells, expressing early developmental genes including RAG1, RAG2 and VPREB1, suggesting the presence of developing B-cells within the tumor. Another B-cell cluster displayed a germinal center-like phenotype, characterized by the expression of BCL6 and AICDA, indicative of local B-cell activation and maturation. These populations were enriched in B2/B3 thymomas, providing molecular evidence for the presence of organized TLSs within the tumor microenvironment. These preliminary findings support a potential link between B-cell organization in thymomas and autoimmune manifestations. To further dissect the spatial architecture and cellular interactions within TLS-like structures, we plan to perform spatial transcriptomic analyses. This approach may provide novel insights into the comprehension of the immunopathogenic mechanisms underlying thymoma-associated autoimmunity.