Current methods for biomarker discovery and target identification in immuno-oncology rely on static snapshots of tumor immunity. To better capture the dynamic and compartmentalized nature of antitumor immune responses, we generated longitudinal “temporal atlases” of productive versus non-productive antitumor immune responses in murine tumor models. We utilized a 34-parameter full spectrum flow cytometry panel to comprehensively profile immune composition within tumors, draining and non-draining lymph nodes, and blood in and around key inflection points of tumor regression or progression. We leveraged two distinct preclinical models for this; the NPK-C1 ectopic prostate cancer model to map dynamics of spontaneous cancer immunoediting, and anti-PD-1 treated MC38 tumors to study response or non-response to immune checkpoint blockade (ICB). We utilized UMAP and FlowSOM algorithms for iterative dimensionality reduction and clustering, respectively, to reveal novel phenotypes associated with productive versus non-productive immunity across model systems, tissues, and time points. We discovered expression of KLRG1 within the intratumoral CD4 T cell compartment was highly associated with tumor progression and response to ICB. Specifically, both FoxP3+ Tregs and FoxP3- Tconv cells within tumors accumulated KLRG1 expression through disease progression, but this was not observed in CD4 T cell or other immune subsets residing in lymph nodes or circulating in blood. Among all intratumoral clusters, KLRG1+ Tconv were the only subset significantly correlated with tumor burden at each time point tested and across both models. KLRG1+ Tconv were significantly enriched in NPK-C1 tumors undergoing progression to escape versus those under immune-mediated equilibrium (p=0.0004) and were lost in animals undergoing curative responses to ICB (p=0.003). In the Treg compartment, unsupervised clustering revealed a KLRG1+Helios- tumor Treg subset that was positively correlated with transition from equilibrium to escape in the NPK-C1 model (p=0.005). Also indicating a potential functional significance, this phenotype was absent in tumors undergoing curative responses to ICB (p=0.0002). Systematic investigation of the functional characteristics, transcriptional programming, and translational significance of intratumoral KLRG1+ CD4 T cell subsets is ongoing. Together, these findings identify KLRG1+ CD4 T cell populations as subsets for further investigation in cancer and demonstrate the utility of longitudinal full spectrum flow cytometry profiling as an engine of dynamic biomarker and/or target discovery in immuno-oncology. Citation Format: Casey Ager, Matthew Chaimowitz, Shruti Bansal, Meri Rogava, Johannes Melms, Catherine Spina, Cory Abate-Shen, Charles G. Drake, Matthew Dallos, Benjamin Izar. KLRG1 marks tumor-infiltrating CD4 T cell subsets associated with immune escape and immunotherapy response. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4115.
Regulatory T cells (Tregs) are highly attractive targets for immunotherapy development, however, there remains a critical need for clinically actionable targeting strategies that specifically inhibit human tumor-infiltrating Tregs (TI-Tregs) while preserving function of cytotoxic effectors and peripheral non-tumor Tregs (P-Tregs). To this end, we sought to identify, validate, and target novel master regulators of TI-Tregs by leveraging a suite of next-generation bioinformatic tools and rigorous ex vivo and in vivo screening and validation methodologies. Specifically, we performed VIPER (Virtual Inference of Protein Activity) analysis on a large dataset of T cell transcriptional profiles from matched peripheral blood and tumors of 36 human patients, yielding 17 master regulator (MR) proteins predicted to uniquely drive the TI-Treg phenotype across cancers. To identify putative therapeutics that modulate TI-Treg MRs, we performed a systematic ex vivo drug screen with an unbiased panel of 1,554 FDA-approved and experimental compounds coupled to RNA sequencing (PLATE-Seq) on human TI-Tregs and P-Tregs. Drug candidates with preferential cytotoxic activity on TI-Tregs versus P-Tregs that also reversed MR transcriptional activity were thoroughly validated in vivo in the MC38 tumor model. In parallel, we performed a pooled in vivo CRISPR/Cas9 screen via the CHIME (CHimeric IMmune Editing) system to identify candidate MRs that regulate TI-Treg recruitment to and/or retention within MC38 tumors. By these approaches, we successfully validated an as-yet poorly described TI-Treg MR, TRPS1 (Transcriptional Repressor GATA Binding 1), which after genetic deletion across the hematopoietic compartment diminished TI-Tregs while preserving P-Tregs in MC38-bearing mice, and led to enhanced spontaneous control of MCA205 sarcomas. In addition, we found the widely used nucleoside analog chemotherapeutic Gemcitabine exhibits preferential inhibitory activity against human TI-Tregs versus P-Tregs, and validated in mice that at sub-clinical dose levels Gemcitabine exhibits immune-dependent therapeutic activity that significantly potentiates checkpoint blockade control of late-stage MC38 tumors. By single cell RNA sequencing of TI- and P-Tregs flow-sorted from Gemcitabine-treated mice, we observe specific depletion of a TI-Treg subset enriched for expression of TI-Treg MRs, including TRPS1. Together, these studies reveal new putative regulators of human TI-Tregs and identify a readily available clinical therapeutic that exhibits inhibitory activity against them. These findings specifically warrant future investigation into the role of TRPS1 in TI-Treg activity and clinical evaluation of low-dose Gemcitabine as a Treg-targeting strategy capable of enhancing checkpoint blockade immunotherapy. Citation Format: Casey R. Ager, Aleksandar Obradovic, Mikko Turunen, Mohsen Khosravi-Maharlooei, Charles Karan, Andrea Califano, Charles G. Drake. Elucidating and targeting master regulators of tumor infiltrating regulatory T cells [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 3612.
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