Abstract B80: High dimensional DNA damage and repair (DDR) cytometric profiling of human T cell subsets revealed distinct DDR signatures
Authors: Muroyama Yuki , Huang Alexander C. , Manne Sasikanth , Herati Ramin S. , Chilukuri Lakshmi , Xu Caiyue , Friedman Claire F. , Zamarin Dmitriy , Wherry E. John 
Published: March 2020

Abstract
DNA damage and repair (DDR) has been extensively studied in the context of inducing cell death of tumor cells. Tumor cells are often highly-proliferating and vulnerable to DNA damage, a foundation for many chemotherapy and radiotherapy approaches. Accumulating evidence indicates the improved clinical prognosis when immune checkpoint blockade (ICB) are combined with chemotherapy or radiotherapy. However, the paradox of the combinational benefit of these proliferation-targeting cytotoxic therapies and proliferation-inducing immunotherapies remains poorly understood. Here, we aimed to interrogate the impact of DNA damage on T cell differentiation and function to better understand the mechanism of the paradox, and to better interrogate how T cell-intrinsic DDR can impact clinical responses to ICBs. We hypothesized that T cell subsets have different DDR responses that contribute to distinct biological outcomes upon exposure to DNA damaging agents and/or proliferative stress induced by ICBs. To test this hypothesis, we developed a novel high dimensional cytometry platform that enables simultaneous analysis of T cell differentiation state and interrogation of multiple DDR pathways at single cell resolution. After introduction of DNA damage by γ-irradiation (IR), UV irradiation (UV) or proliferative stress (i.e. aCD3/28 stimulation) DNA damage accumulation and response varied depending on the state of T cell differentiation. For example, terminally differentiated effector cells (TEMRA) had significantly higher DNA damage accumulation and cell death. In contrast, stem cell memory (TSCM) and regulatory T cells (Treg) displayed high DDR with less cell death, suggesting perhaps better cell-intrinsic DDR against genotoxic stress. Overall, this platform revealed consistent, T cell subset specific patterns of DDR as well as specific DDR pathways induced by different types of DNA damage. DDR-Immune profiling of PBMC from uterine cancer patients treated with the combination of chemotherapy and ICB indicated the correlation between distinct patterns of DDR-Immune signatures and therapeutic response. Notably, in clinical responders, Ki67+ CD8 T cells responding to PD-1 blockade had increased pATM, perhaps in response to proliferative stress induced by PD-1 blockade. This increase in pATM was not observed in clinical non-responders. In addition, Treg from clinical non-responders had elevated DDR responses compared to responders, suggesting the possibility of greater resistance to genotoxic/proliferative stress. Collectively, our new platform shed lights on previously unrecognized roles of T cell-intrinsic DDR as a novel determinant of T cell differentiation and immune responsiveness. These findings may have relevance to apply to understanding the paradox of synergy of chemotherapy and radiotherapy with ICBs.
Citation Format: Yuki Muroyama, Alexander C. Huang, Sasikanth Manne, Ramin S. Herati, Lakshmi Chilukuri, Caiyue Xu, Claire F. Friedman, Dmitriy Zamarin, E. John Wherry. High dimensional DNA damage and repair (DDR) cytometric profiling of human T cell subsets revealed distinct DDR signatures [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B80.