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On one hand, regulatory T cells (Tregs) play an immunosuppressive activity in most solid tumors but not all. On the other hand, the organization of tumor-infiltrating immune cells into tertiary lymphoid structures (TLS) is associated with long-term survival in most cancers. Here, we investigated the role of Tregs in the context of Non-Small Cell Lung Cancer (NSCLC)-associated TLS. We observed that Tregs show a similar immune profile in TLS and non-TLS areas. Autologous tumor-infiltrating Tregs inhibit the proliferation and cytokine secretion of CD4⁺ conventional T cells, a capacity which is recovered by antibodies against Cytotoxic T-Lymphocyte-Associated protein-4 (CTLA-4) and Glucocorticoid-Induced TNFR-Related protein (GITR) but not against other immune checkpoint (ICP) molecules. Tregs in the whole tumor, including in TLS, are associated with a poor outcome of NSCLC patients, and combination with …