Abstract A026: Lysosomal ion gradients are key determinants of T cell metabolism and function

Abstract We discovered that high extracellular potassium (↑[K+]e) within tumors limits T cell effector function and exhaustion. Moreover, the existing model of K+ transport in T cells could not account for our results. Specifically, ↑[K+]e constrained T cell activation and exhaustion independent of its impact upon membrane potential (Vm) or Ca2+ influx. Rather, ↑[K+]e controlled T cell state by elevating intracellular K+ ([K+]i), limiting Akt-mTOR signaling and inducing a state of autophagy despite exogenous nutrient sufficiency (Eil Nature 2016 & Science 2019). Thus, T cell ion transport is relevant to antitumor function but poorly understood. To apply ionic reprogramming as a means to advance cancer immunotherapy, we performed an in vivo CRISPR Cas9 screen targeting ion transporters we found to be expressed across T cell state. We transduced spCas9 OT-I TCR transgenic CD8+ T cells with retroviral particles encoding a custom pool of sgRNAs targeting ion transporters expressed in CD8 T cells (133 ion transporters with 6 sgRNAs per target) that we then transferred to B16 (murine melanoma)-OVA bearing mice. Our analyses identified numerous transmembrane and organellar ion transporters not previously recognized to have a role in T cell behavior as critical regulators of T cell function. Those transporters whose genetic disruption impacted T cell persistence in tumors converged on maintenance cytoplasmic K+ concentration and organellar transporters of Na+, H+, and K+. The importance of cytoplasmic K+ was apparent in the impact of Atp1a1 disruption (α subunit of the Na+/K+ ATPase), resulting in a severe defect of persistence in both primary tumor and lymphoid sites. The provision of exogenous K+ restored intracellular levels in Atp1a1-deficient T cells and prevented the accumulation of toxic metabolites (ROS; Collier & Eil, Cancer Immunol Res 2023). Conversely, enhanced Na+/ K+ ATPase expression and ↑[K+]e increased the in vivo persistence of CD8 T cells in vivo. The impact of numerous additional sgRNAs from our screen highlighted the previously unrecognized importance of endolysosomal transporters of Na+, H+, and K+ in T cell function within tumors. Moreover, ongoing proteomic analysis of CD8+ T cells in ↑[K+]e also highlights the relationship between endolysosome metabolism balance. These findings encourage a reconsideration of cytoplasmic and endolysosomal ion gradients in cellular metabolism to inform novel therapeutic strategies targeting T cell ion transport in tumors. Additional translational relevant questions include the impact of K+ gradients within tumors upon the function of other types of immune cells. Citation Format: Chelsea Jenkins, Jessica Briones, Robert MD Eil. Lysosomal ion gradients are key determinants of T cell metabolism and function [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2024 Oct 18-21; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2024;12(10 Suppl):Abstract nr A026.