Abstract PO032: TIM3 regulation by phosphatidylserine

Abstract

Abstract Immune responses have been successfully reactivated in cancer by targeting inhibitory receptors such as PD-1 on exhausted T cells to block immune checkpoints. Many patients do not respond, however, and a key challenge in immuno-oncology is to identify and understand new immune-regulatory targets to increase response rates. One other co-inhibitory receptor in T cells is TIM3, which – like PD-1 – is upregulated on exhausted T cells in the tumor microenvironment. TIM3 belongs to a family of phosphatidylserine (PS) receptors with well-documented roles in efferocytosis by phagocytes. However, the role of PS in regulating TIM3 remains unclear. Previous studies have shown that TIM3 can variously enhance or inhibit T cell signaling, though exactly how TIM3 accomplishes this range of activity is unknown. We therefore investigated how TIM3 levels modulate T cell signaling, and how PS in turn influences TIM3 activity. We found that TIM3 promoted T cell activity in a Jurkat T cell model when expressed at high – but not low – levels on the cell surface. We also confirmed the ability of human TIM3 to bind PS with low micromolar affinity using surface plasmon resonance. To investigate how PS affects the ability of TIM3 to promote T cell signaling in this model, we employed several approaches, including adding PS to the cell culture, manipulating TIM3 to reduce or abrogate PS binding (using chimeric receptors and TIM3 mutants), and pharmacological blockade of PS binding using a functional TIM3 antibody. Together, these studies supported a role for PS in regulating TIM3 activity, where decreased or abrogated PS binding prevented TIM3 from promoting T cell signaling. We also sought to understand what changes in T cell receptor (TCR) signaling might account for the TIM3-dependent effects we observed. We found that TIM3 did not impact phosphorylation of downstream components in the TCR signaling cascade. Rather, cells expressing high – but not low – surface levels of TIM3 displayed enhanced phosphorylation of receptor components in the TCR signaling pathway, similar to what has been reported for PD-1. We hypothesize that TIM3 modulates TCR signaling by modifying the localization and/or dwell time of one or more signaling effector proteins (e.g. phosphatases) in a manner dependent on PS engagement. The consequences of this are then determined by surface levels of TIM3. Our results demonstrate that PS does function as a ligand capable of modulating TIM3 activity, and provide new mechanistic insight into how TIM3 modulates TCR signaling. Citation Format: Courtney M. Smith, Alice Li, Nithya Krishnamurthy, Mark A. Lemmon. TIM3 regulation by phosphatidylserine [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO032.