Background Regulatory T cells (Treg) -targeted therapy exhibit clinical benefit and has been reported as a promising strategy. However, many gaps remain in our understanding of Treg biology within the context of tumor microenvironment (TME). The autoimmune toxicity and restricted efficacy are major limitations of Treg therapies in the clinic, when Treg depletion occurred not only in the tumor but in other organ systems, or concurrent downregulation of antitumor effector T cells.1, 2
Methods We profiled 51,195 single-cell transcriptomes of CD4+ T cells in tumors and peripheral blood from patients with head and neck squamous cell carcinomas (HNSCC)3, in inflamed tonsil tissues and in healthy peripheral blood. Canonical genes, gene sets and RNA Velocity4 were used to define cell states of Treg. Cibersortx5 and bulk RNA sequencing data in The Cancer Genome Atlas were used to infer the association between the enrichment of Treg subpopulations and progression-free survival of patients with solid tumors. SCENIC6 and Causal mixed graphical modeling7 were used to reconstruct the gene regulatory network (GRN). Knockout of BATF with CRISPR-Cas98 in conjunction with bulk RNA sequencing, immunophenotyping and in vitro functional assays were used to interrogate the roles of BATF in human activated Treg.
Results We identified an activated subpopulation of Treg expressing multiple tumor necrosis factor receptor (TNFR) genes, including OX40 and 4-1BB, which is highly enriched in solid TME compared with non-tumor tissues. These TNFR-activated Treg were associated with worse prognosis across solid tumors. Notably, we found BATF is a central component of a GRN that controls the transcriptional signature of TNFR-activated Treg. Consistent with single-cell analyses, BATF was co-expressed with 4-1BB, OX40, CD96 and CD39 that highly enriched in HNSCC intratumoral Treg at protein level. CRISPR-editing results revealed an enhancement of immunosuppression in BATF KO Treg and activation in BATF KO Treg accompanied with increased expression of genes including 4-1BB, OX40, ICOS, LAG3 and neuropilin-1, indicating that BATF functions as a transcriptional nexus in human activated Treg that essential for Treg activation, function and stability.
Conclusions We identify a unique intertumoral subpopulation of Treg characterized by BATF-driven expression of tumor necrosis factor receptor family expression and associated with survival across solid tumors, suggesting a possibility to target suppressive intratumoral Treg without causing overt autoimmunity in normal tissues. A deeper understanding of transcriptional network in Treg biology will provide novel mechanisms for immunotherapies in cancer, but also for Treg engineering in autoimmunity.
Acknowledgements We thank the Vignali, Bruno and Benos Labs for all their constructive comments and feedback.
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Ethics Approval All patients provided informed written consent, and this study was approved by our Institutional Review Board (University of Pittsburgh Cancer Institute, Tissue Collection Protocol 99-069).
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