Dykema, Arbor G., Zhang, Jiajia, Cheung, Laurene S., Connor, Sydney, Zhang, Boyang, Zeng, Zhen, Cherry, Christopher M., Li, Taibo, Caushi, Justina X., Nishimoto, Marni, Munoz, Andrew J., Ji, Zhicheng, Hou, Wenpin, Zhan, Wentao, Singh, Dipika, Zhang, Tianbei, Rashid, Rufiaat, Mitchell-Flack, Marisa, Bom, Sadhana, and Tam, Ada
Regulatory T cells (Treg) are conventionally viewed as suppressors of endogenous and therapy-induced antitumor immunity; however, their role in modulating responses to immune checkpoint blockade (ICB) is unclear. In this study, we integrated single-cell RNA-seq/T cell receptor sequencing (TCRseq) of >73,000 tumor-infiltrating Treg (TIL-Treg) from anti–PD-1–treated and treatment-naive non–small cell lung cancers (NSCLC) with single-cell analysis of tumor-associated antigen (TAA)–specific Treg derived from a murine tumor model. We identified 10 subsets of human TIL-Treg, most of which have high concordance with murine TIL-Treg subsets. Only one subset selectively expresses high levels of TNFRSF4 (OX40) and TNFRSF18 (GITR), whose engangement by cognate ligand mediated proliferative programs and NF-κB activation, as well as multiple genes involved in Treg suppression, including LAG3. Functionally, the OX40hiGITRhi subset is the most highly suppressive ex vivo, and its higher representation among total TIL-Treg correlated with resistance to PD-1 blockade. Unexpectedly, in the murine tumor model, we found that virtually all TIL-Treg–expressing T cell receptors that are specific for TAA fully develop a distinct TH1-like signature over a 2-week period after entry into the tumor, down-regulating FoxP3 and up-regulating expression of TBX21 (Tbet), IFNG, and certain proinflammatory granzymes. Transfer learning of a gene score from the murine TAA-specific TH1-like Treg subset to the human single-cell dataset revealed a highly analogous subcluster that was enriched in anti–PD-1–responding tumors. These findings demonstrate that TIL-Treg partition into multiple distinct transcriptionally defined subsets with potentially opposing effects on ICB-induced antitumor immunity and suggest that TAA-specific TIL-Treg may positively contribute to antitumor responses. Editor's summary: Immune checkpoint blockade (ICB) has limited success in non–small cell lung cancer (NSCLC) patients. ICB activates T cells to drive tumor killing; however, anti-tumor responses are often dampened by immunosuppressive mechanisms. Dykema et al. investigated how regulatory T cells (Treg) contribute to anti-tumor immunity during anti-PD-1 treatment. By integrating single cell TCRseq/RNAseq from human NSCLC patients and murine tumors, multiple tumor Treg subclusters were identified. An "activated" subcluster expressing TNFR superfamily genes OX40 and GITR was highly suppressive and associated with ICB resistance. In murine tumors the majority of tumor-reactive Treg differentiated into a proinflammatory Th1-like phenotype, and this Th1-like subcluster was enriched in human anti-PD-1–responsive lung tumors. These findings identify diversity within tumor Treg and suggest that targeting specific subclusters could improve responses to ICB. —Hannah Isles [ABSTRACT FROM AUTHOR]