We thank K Maekawa-Kato for technical assistance

We thank K Maekawa-Kato for technical assistance. cells in TETs, we investigated not only cytokine production by T cells, but also their cytotoxicity using bispecific T-cell engager technology. The cluster analysis of T-cell profiles based on flow cytometric data revealed that type B3 thymoma and thymic carcinoma (B3/C) belonged to the hot cluster characterized by a high proportion of Tim-3+ and CD103+ in CD4 and CD8 single-positive T cells. Enhancements in cytokine production and the cytotoxicity of T cells by the anti-PD-1 antibody were significantly greater in B3/C. These results indicate the potential of immunotherapy for patients with B3/C. and models make it difficult to develop standard treatments. Complete surgical resection is reportedly the only chance for a cure in TETs4,5. However, even after complete resection, the recurrence rates of type B3 and type C thymoma (thymic carcinoma) are 27 and 50%, respectively6. Surgery cannot be indicated for some patients when tumors invade the surrounding organs, such as the heart and great vessels, and metastasize to multiple organs. More aggressive histological types of TETs often present at an advanced stage and result in worse overall survival. Tioconazole Chemotherapy, radiation therapy, and molecular-targeting agents are also options in combinatorial treatment strategies7,8. Immune checkpoint inhibitors began a new era in cancer immunotherapy. The anti-PD-1 blocking Tioconazole antibody exerts beneficial effects in a limited population of cancer patients9. Tioconazole Indications for the anti-PD-1 blocking antibody are expanding and now include TETs. Clinical trials on immune checkpoint inhibitors RPTOR are ongoing, and acceptable clinical efficacies of the anti-PD-1 antibody have been reported for TETs10,11. In the development of anti-PD-1 therapy for TETs, it is crucial to establish a method that identifies target patients who are more likely to respond to the drug. Therefore, it is important to have a clear understanding of the tumor immune microenvironment of TETs. However, the lack of and models makes it difficult to study the tumor immune microenvironment of TETs. The method currently available for the classification of TETs is the WHO histopathological classification, which is Tioconazole based on the morphology of epithelial tumor cells and the proportion of intratumor lymphocytic involvement. The majority of intratumor lymphocytes of TETs are CD4+CD8+ double-positive T cells, which are Tioconazole undifferentiated and functionally immature T cells. On the other hand, CD4 or CD8 single-positive T cells play major roles in cancer immunology. However, the roles of CD4 and CD8 single-positive T cells in TETs have not yet been elucidated in detail from the aspect of cancer immunology. Therefore, in the present study, we focused on the phenotypic and functional properties of CD4 and CD8 single-positive T cells in surgically resected TETs as a step towards establishing a rationale for immunotherapy for TETs. Results Clinicopathological findings The clinical and pathological features of patients with TETs are summarized in Table?1. Thirty-one cases of TETs included 10 males (32%) and 21 females (68%) with a mean age of 58 years old (range: 36C82). Thymic carcinoma included 4 squamous cell carcinomas and 2 lymphoepithelioma-like carcinomas. Four patients had a medical history of myasthenia gravis (MG). Three of these patients were diagnosed with type B1 thymoma, and acetylcholinesterase inhibitors were administered to control MG symptoms. The remainder of patients were diagnosed with type B2 thymoma without medication for MG. One patient diagnosed with type AB thymoma had a medical history of pure red cell aplasia (PRCA), and cyclosporine was administered preoperatively to control anemia. Table 1 Patient characteristics. stimulation for intracellular cytokine staining Freshly isolated cells or CD4 single-positive T cells purified by the FACS Aria II cell sorter (BD Biosciences) from TET tissues were stimulated with 50?ng/ml phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich), 1?g/ml ionomycin (Sigma-Aldrich), and Golgi Stop reagent (BD Biosciences) at 37?C, 5% CO2, for 5?hours. Harvested cells were washed and stained with antibodies against surface antigens and fixable viability dye (eBioscience) at 4?C for 20?minutes. After the incubation, cells were washed, fixed, and permeabilized with Cytofix/Cytoperm solution (BD Bioscience) at 4?C for 30?minutes. Intracellular cytokines (IFN-, TNF-, and IL-2) were then stained with antibodies for IFN- (clone 4SB3; Biolegend), TNF- (clone MAb11; Biolegend), and IL-2 (clone MQ1-17H12; eBioscience), followed by FACS analyses. T-cell cytotoxicity assay The.