Abstract 4115: KLRG1 marks tumor-infiltrating CD4 T cell subsets associated with immune escape and immunotherapy response
Authors: Ager Casey , Chaimowitz Matthew , Bansal Shruti , Rogava Meri , Melms Johannes , Spina Catherine , Abate-Shen Cory , Drake Charles G. , Dallos Matthew , Izar Benjamin 
Published: April 2023

Abstract
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.