Single-cell identifies and validates human circulating Treg subtype/state Treg<sup>fci</sup> in non-small cell lung cancer

Regulatory T cells (Treg) play immunosuppressive roles in lung cancer and are key biomarkers in cancer immunology. Using scRNA-seq and stereo-seq, we aimed to identify a novel circulating Treg subtype in non-small cell lung cancer (NSCLC). We developed a regional overlap-expression rate (rOER) system with cell identity marker gene panels (ciMGPs) to quantify T cell subtype specificity across pre/post-operative blood, 31 diseases, and in 17 organs. A Treg subtype (Treg^fci, FOXP3⁺CTLA4⁺IL2RA⁺) was identified and validated for disease-, organ-, and time-specific characteristics across spatializations, temporalizations, diseases, and prognoses. Treg^fci was prominently enriched in pre-operative blood and NSCLC tissue but markedly decreased or was absent post-operatively. Spatial transcriptome and multiplex immunostaining revealed that Treg^fci localized primarily to the interface between NSCLC and normal tissues, as well as in specific micro-niches. ETS Proto-Oncogene 1 (ETS1) was upregulated in Treg^fci, confirmed by knockdown and knockout experiments. ETS1 facilitated Treg^fci migration from circulation into tumor tissues via cancer cell-derived chemoattraction. Interactions between ETS1 and Treg^fci ciMGPs, and between intracellular organelles, were found to modulate metabolism and mitochondrial function. These findings highlight the importance of Treg^fci in understanding the molecular mechanism by which Tregs maintain the balance between systemic and local immune ecosystems. ETS1's regulation of Treg^fci transcriptomic and metabolic profiles were confirmed in human PBMCs treated with an ETS1 inhibitor and in lung tissues from mice^ETS1-cKO mice. Furthermore, Treg^fci and ETS1 represent a distinct molecular signature of T cells and potential therapeutic targets for clinical intervention in lung cancer.