TIM3 is a context-dependent coregulator of cytotoxic T cell function

Abstract

Cytotoxic T lymphocytes (CTLs) are essential effectors in the antiviral and antitumour immune response and attractive targets in cancer immunotherapy. Although CTLs can directly recognise and kill tumour cells, CTLs become suppressed in the tumour microenvironment. This project investigated the inhibitory receptor T cell immunoglobulin and mucin domain 3 (TIM3). TIM3 is expressed on T cells after chronic antigen exposure and marks the most exhausted tumour infiltrating CTLs in multiple solid tumours. However, it is unclear whether TIM3 directly regulates CTL function. In addition, despite its predominantly inhibitory role in vivo, TIM3 can promote cellular activation in T and non-T cells, and the roles of putative ligands in TIM3 function are disputed. Therefore, we aimed to determine the effect of TIM3 on direct CTL antitumour function and how the TIM3 ligands Galectin9 (GAL9) and CEACAM1 regulate its function. We employed three-dimensional (3D) tumour spheroids that effectively induce CTL suppression similar to the in vivo tumour microenvironment in comparison to conventional two-dimensional (2D) tumour cell culture. In the 3D spheroid model, TIM3 significantly inhibited CTL cytotoxicity and cytoskeletal polarisation as a key mechanism of effective cytolysis in murine and human CTLs. In contrast, in the 2D tumour model, TIM3 stimulated CTL cytotoxicity, cytoskeletal polarisation, and secretion of the immune-stimulatory cytokine interferon γ (IFNγ). Expression of GAL9 and CEACAM1 in trans on tumour cells further suppressed the CTL killing ability in the 3D spheroid model and enhanced costimulatory function in 2D. CEACAM1 in cis neutralised TIM3 functions in both 3D and 2D. We suggest that TIM3 functions as a context-dependent coregulatory receptor, as supported by the engagement of its ligands GAL9 and CEACAM1. In a largely stimulatory signalling context of a CTL, TIM3 functions as a costimulator, and in a more inhibitory context, TIM3 functions as a coinhibitor.