Characterization of tumor-associated T cells in patients with thymic epithelial tumors

Thymic epithelial tumors (TETs), including thymomas and thymic carcinomas (TCs), are heterogeneous and rare tumors that originate from thymic epithelial cells (TECs). They represent the most common primary neoplasm of the anterior mediastinum, with an overall annual incidence of 0.15/100.000 individuals. The typical presentation is around the 5th and 6th decades of life, without gender predilection. Complete surgical resection is the gold standard therapy for TETs; yet, standard treatment after first-line failure is still lacking, mainly due to the rarity and heterogeneity of the disease1,2. According to the 2015 World Health Organization (WHO) classification, TETs are classified based on the histological appearance of thymic epithelial cells and the proportions of lymphocytes. They are divided into 6 subtypes, including A, AB, B1, B2, B3, and C (the latest corresponding to TCs), with a progressive worsening of the prognosis as the lesion progresses from A to C3. Thymomas and thymic hyperplasia (TH), which is a benign form of thymic epithelial lesions, have been associated with paraneoplastic autoimmune syndromes more frequently than have TCs. In particular, thymomas are strongly associated with myasthenia gravis (MG), caused by the production of autoantibodies targeting neuromuscular junction proteins and occurring in up to 45% of thymoma patients4. The pathophysiological links between thymoma and autoimmunity still need to be elucidated, however, a possible explanation could rely on the intratumoral thymopoiesis that has been reported in these lesions5. Indeed, the alteration of the thymic architecture, together with functional defects of TECs, can affect central tolerance leading to the release of autoreactive T cells in the peripheral blood. Despite evidence suggesting a role for T lymphocytes in the immune dysregulation of TETs, the differential roles of tumor-infiltrating T cells in the different TET subtypes have still to be defined. Yet, their comprehension may guide the treatment of TET patients upon failure of first-line surgical resection. Therefore, in this study we characterized T cells in patients with different types of thymic epithelial lesions. 22 patients (5 THs; 4 A, 6 AB and 4 B3 thymomas; 3 TCs) undergoing thymic surgery were enrolled by the Humanitas Unit of Thoracic Surgery. Pediatric patients undergoing thymectomy for congenital heart defects were included as healthy controls (HCs). T cells were characterized by flow cytometry, using an 18-color panel that allowed us to follow the maturation of thymocytes (CD3, TCR, CD4, CD8, CD27, CCR7, CD45RO, CD57, CD69), to investigate the presence of regulatory T cells (Tregs) (CD127, CD25, TNFR2, OX40), and to assess the activation state of T cells (OX40, CD95) and their expression of immune checkpoints (PD1, TIM3). Our results indicated that TH and type AB thymomas, and to a lesser extent type B3 thymomas, are characterized by an active thymopoiesis that partially recapitulated thymopoiesis occurring in pediatric thymuses. In particular, type AB thymomas showed important aberrancies in the thymopoietic process, characterized by a significant increase in the frequency of CD4+ immature single positive (CD4ISP) thymocytes, an intermediate thymocyte population that still lacks CD3 expression. Type AB thymomas were also characterized by an increased ratio of double-positive (DP) over single-positive (SP) thymocytes, likely sustained by the significant reduction of CD69 expression on DP thymocytes observed in these patients. Thymopoiesis was negligible in TCs, which were rather characterized by a significant increase in the frequency of mature CD4+ T lymphocytes. These cells were composed for up to 30% of CD25highCD127low Tregs that showed a high expression of the immune checkpoints PD1 and TIM-3 (detected on up to 80% and 60% of Tregs, respectively). Notably, the TNFR2+/OX40+ subpopulation of Tregs, which is known to exert maximal suppressive activity6, was also enriched in TCs. By demonstrating profound differences among T cells infiltrating different types of TETs, the results of this study may provide important insights into the comprehension of the reciprocal interactions between tumoral TECs and T cells and pave the way for a personalized therapeutic approach to TET patients.