Diversity of intratumoral regulatory T cells in B-cell non-Hodgkin lymphoma.

Tumor-infiltrating regulatory T cells (Tregs) contribute to an immunosuppressive tumor microenvironment. Despite extensive studies, the prognostic impact of tumor-infiltrating Tregs in B-cell non-Hodgkin lymphomas (B-NHL) remains unclear. Emerging studies suggest substantial heterogeneity in the phenotypes and suppressive capacities of Tregs, emphasizing the importance of understanding Treg diversity and the need for additional markers to identify highly suppressive Tregs. Here, we applied single-cell RNA sequencing and TCR sequencing combined with high-dimensional cytometry to decipher the heterogeneity of intratumoral Tregs in diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL), compared with non-malignant tonsillar tissue. We identified three distinct transcriptional states of Tregs; resting, activated and unconventional LAG3+FOXP3- Tregs. Activated Tregs were enriched in B-NHL tumors, co-expressed several checkpoint receptors and had stronger immunosuppressive activity compared with resting Tregs. In FL, activated Tregs were found in closer proximity to CD4+ and CD8+ T cells than other cell types. Furthermore, we used a computational approach to develop unique gene signature matrices which were used to enumerate each Treg subset in cohorts with bulk gene expression data. In two independent FL cohorts, activated Tregs was the major subset, and high abundance was associated with adverse outcome. This study demonstrates that Tregs infiltrating NHL tumors are transcriptionally and functionally diverse. Highly immunosuppressive activated Tregs were enriched in tumor tissue but absent in peripheral blood. Our data suggest that a deeper understanding of Treg heterogeneity in B-NHL could open new paths for rational drug design, facilitating selective targeting to improve anti-tumor immunity.