Flow cytometric characterization of αβ and γδ T cells in patients with thymic epithelial lesions

Thymic epithelial lesions (TELs) are rare tumors of the anterior mediastinum that arise from thymic epithelial cells (TECs). TELs are subdivided according to the World Health Organization classification into thymic hyperplasia (TH), thymoma (Ty), and thymic carcinoma (TC). Based on the cellular composition, the morphology of the tumor epithelial cells, and the tumor architecture, thymomas are further subdivided into A, AB, B1, B2, and B3 types, with a worse prognosis as the lesion progresses from type A to B3, and the worst prognosis in TC. TELs, in particular TH and Tys, are characterized by two peculiar features: an active intratumor thymopoiesis and the association with autoimmune disorders (ADs), with Myasthenia Gravis (MG) being the most common, occurring in up to 40% of thymoma patients. It is likely that the active intratumor thymopoiesis, together with alterations of the thymic architecture and functional defects of TECs reported in these patients may affect mechanisms of central tolerance leading to the release of autoreactive T cells in the peripheral blood. However, the precise pathophysiological link between TELs and ADs still need to be elucidated. Therefore, in this study, we characterized tissue-associated and circulating αβ and γδ T cells in 25 patients affected by different TEL types (7 THs; 4 A, 7 AB, and 4 B3 Tys; 3 TCs) undergoing thymic surgery at the Humanitas Unit of Thoracic Surgery. In addition, cells obtained from thymic samples of 6 pediatric patients undergoing cardiac surgery for cardiac defects, and peripheral blood mononuclear cells (PBMCs) obtained from 7 age- and sex-matched healthy donors were used as healthy counterparts of tumor-associated and circulating T cells, respectively. αβ and γδ T cells were characterized by using two distinct flow cytometric panels that allowed us to follow the maturation of thymocytes and assess the expression of activating and inhibitory molecules. Data were analyzed using both manual gating strategy and PhenoGraph, an unsupervised and unbiased algorithm that automatically identifies clusters composed by phenotypically identical cells. TH, Type AB and B3 Tys were characterized by an active thymopoiesis as indicated by the presence of immature thymocytes states, such as CD4 immature single-positive cells and double-positive thymocytes. Moreover, type AB Tys were also enriched in Vδ1 T cells, suggesting that active thymopoiesis was not restricted to the αβ T cell lineage. In addition, type AB Tys displayed a particular thymocyte population characterized by the expression of CD3, CD4, CD8, and Vδ1, which, specifically in these tumors, may suggest an alteration in the T cell lineage determination. Vδ1 T cell frequency was also significantly higher in the peripheral blood of type AB Tys compared with HD, suggesting that type AB Tys may actively export Vδ1 T cells in the periphery. Notably, circulating Vδ1 T cells of patients with type AB Tys and TH were characterized by a high expression of CD27, a marker that may suggest their naïveness and therefore their thymic origin. On the contrary, αβ T cells were not increased in the blood of these patients, suggesting a possible impairment in the maturation of these cells. Finally, TC showed a high frequency of circulating Vδ1 T cells likely not deriving from the tumor, as TCs did not show an active thymopoiesis. Notably, TC patients were characterized by the presence of two Vδ1 T cell clusters that were not observed neither in HDs nor in TH and Ty patients. These clusters were characterized by a high expression of TIGIT, CD16, CD56, and CD158b1b2j indicating a terminally differentiated effector cell phenotype. Interestingly, one of the two clusters lacked the expression of the senescence marker CD57, while expressing high levels of CD69, a marker of tissue residency and early activation, suggesting a possible recirculation of these cells from the tumor into the periphery maintaining an activated profile. Altogether, our results demonstrate the presence of an active intratumor thymopoiesis in TH and type AB Tys that appear to be more functional for the γδ T cell lineage. The role played by Vδ1 T cells in TCs, in the periphery and within the tumor tissue, may deserve further investigation to understand the possible role of these cells in the control of tumor progression and patients’ overall survival.