Background Regulatory T cells (T_reg) -targeted therapy exhibit clinical benefit and has been reported as a promising strategy. However, many gaps remain in our understanding of T_reg biology within the context of tumor microenvironment (TME). The autoimmune toxicity and restricted efficacy are major limitations of T_reg therapies in the clinic, when T_reg 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 T_reg. Cibersortx⁵ and bulk RNA sequencing data in The Cancer Genome Atlas were used to infer the association between the enrichment of T_reg 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 T_reg.

Results We identified an activated subpopulation of T_reg 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 T_reg 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 T_reg. Consistent with single-cell analyses, BATF was co-expressed with 4-1BB, OX40, CD96 and CD39 that highly enriched in HNSCC intratumoral T_reg at protein level. CRISPR-editing results revealed an enhancement of immunosuppression in BATF KO T_reg and activation in BATF KO T_reg 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 T_reg that essential for T_reg activation, function and stability.

Conclusions We identify a unique intertumoral subpopulation of T_reg 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 T_reg without causing overt autoimmunity in normal tissues. A deeper understanding of transcriptional network in T_reg biology will provide novel mechanisms for immunotherapies in cancer, but also for T_reg engineering in autoimmunity.

Acknowledgements We thank the Vignali, Bruno and Benos Labs for all their constructive comments and feedback.

References

1. Zappasodi R, Sirard C, Li Y, Budhu S, Abu-Akeel M, Liu C, et al. Rational design of anti-GITR-based combination immunotherapy. Nature Medicine 2019;25(5):759–66.

2. Jacobs JF, Punt CJ, Lesterhuis WJ, Sutmuller RP, Brouwer HM, Scharenborg NM, et al. Dendritic cell vaccination in combination with anti-CD25 monoclonal antibody treatment: a phase I/II study in metastatic melanoma patients. Clin Cancer Res 2010;16(20):5067–78.

3. Cillo AR, Kürten CHL, Tabib T, Qi Z, Onkar S, Wang T, et al. Immune Landscape of Viral- and Carcinogen-Driven Head and Neck Cancer. Immunity 2020;52(1):183–99.

4. Bergen V, Lange M, Peidli S, Wolf FA, Theis FJ. Generalizing RNA velocity to transient cell states through dynamical modeling. Nature Biotechnology 2020;38(12):1408–14.

5. Newman AM, Steen CB, Liu CL, Gentles AJ, Chaudhuri AA, Scherer F, et al. Determining cell type abundance and expression from bulk tissues with digital cytometry. Nature Biotechnology 2019;37(7):773–82.

6. Van de Sande B, Flerin C, Davie K, De Waegeneer M, Hulselmans G, Aibar S, et al. A scalable SCENIC workflow for single-cell gene regulatory network analysis. Nature Protocols 2020;15(7):2247–76.

7. Sedgewick AJ, Buschur K, Shi I, Ramsey JD, Raghu VK, Manatakis DV, et al. Mixed graphical models for integrative causal analysis with application to chronic lung disease diagnosis and prognosis. Bioinformatics 2019;35(7):1204–12.

8. Schumann K, Raju SS, Lauber M, Kolb S, Shifrut E, Cortez JT, et al. Functional CRISPR dissection of gene networks controlling human regulatory T cell identity. Nature Immunology 2020;21(11):1456–66.

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).