Abstract 595: Distinct tumor infiltrating Treg lineages are associated with response to anti-PD1 checkpoint blockade in NSCLC

Abstract Immune-checkpoint blockade (ICB) has proved a major success, especially in highly mutated tumors such as lung cancer. Nevertheless, not all patients respond to ICB. It is possible that regulatory T cells (Tregs) play a role in this lack of response by suppressing tumor-reactive cytotoxic T cells, however the specific mechanisms that lead to this suppression remain elusive. It is therefore necessary to understand the functional programming and suppressive nature of Treg subsets in the tumor microenvironment to define targetable molecules for future biomarker-driven therapeutics. In this study we performed single cell RNA-sequencing on T cells isolated from resected tissue and peripheral blood from 15 neoadjuvant nivolumab (anti-PD1)-treated and 10 untreated non-small cell lung cancer (NSCLC) patients. We identified and analyzed 71,251 CD4+ FoxP3+ Tregs. Refined clustering was performed, and we used pseudotime and differential gene analyses to understand the transcriptional relationship between clusters and patient groups. With our highly refined clustering approach, we identified 8 separate Treg clusters that reflect differing functionalities within the tumor microenvironment. We demonstrate distinct Treg subsets that diverge towards either an activated state, expressing members of the tumor necrosis factor receptor (TNFR) superfamily: OX40, 41BB, GITR, or a resting state. Patients who respond to ICI have a decreased activated Treg score and demonstrate RNA velocity trajectory from activated Tregs towards more in-active and resting populations. Untreated patients conversely show a high activated Treg score with RNA velocity demonstrating activated Tregs are a terminal differentiation state. We hypothesize that ICI treatment pushes Tregs away from an activated phenotype towards more quiescent and that the efficiency of this transition may predict response to ICI. We plan to stimulate receptors associated with non-response using TNFR agonist ligands and hypothesize that their induced signaling will result in transcriptional program changes altering the suppressive ability of Tregs. In addition, we show that Tregs who experience antigen within the tumor microenvironment are more suppressive than bystander Tregs that home to the tumor without antigen stimulation. Together, this study provides an in-depth look at the Treg-derived suppressive mechanisms governing their function in the TME of anti-PD-1-treated vs. untreated tumors. This in-depth analysis of tumor Tregs has identified specific targetable biomarkers which could be used to improve ICB response while mitigating off-target immune adverse events by specifically inhibiting a small subset of Tregs without disturbing systemic immune homeostasis. Citation Format: Arbor G. Dykema, Jiajia Zhang, Boyang Zhang, Taibo Li, Justina X. Caushi, Laurene S. Cheung, Hongkai Ji, Zhicheng Ji, Kellie N. Smith, Drew M. Pardoll. Distinct tumor infiltrating Treg lineages are associated with response to anti-PD1 checkpoint blockade in NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 595.