Date of Award
Doctor of Philosophy (PhD)
TIM-3, or T cell, immunoglobulin, and mucin domain-containing protein-3, is a transmembrane glycoprotein expressed by several types of immune cells, including T cells, making it an important regulator of the immune response. However, the biology of TIM-3 is complicated, as it has been shown to variously promote and inhibit the function of T cells and other immune cells. Understanding the contexts in which TIM-3 serves these dual roles has remained difficult, as the ligands that regulate the activity of this receptor are still unclear. Several molecules have been proposed to regulate TIM-3, including phosphatidylserine (PS), galectin-9, carcinoembryonic antigen-related cell adhesion molecules 1 (CEACAM1), and high motility group box 1 (HMGB1). The majority of the literature has focused on galectin-9, a bivalent glycan-binding molecule with additional binding partners, making it unclear if galectin-9 has TIM-3-specific effects. Phosphatidylserine, on the other hand, is of particular interest, despite its lack of attention in the literature. The TIM family are known as PS receptors with important roles in recognizing and clearing apoptotic cells. Previous studies confirm the ability of TIM-3 to bind PS, but additional studies on its ability to modulate TIM-3 function, beyond phagocytosing apoptotic cells, are lacking. The main goal of my dissertation was to determine how ligand regulation, in particular, PS-binding, contributes to TIM-3 function. I also investigated how galectin-9 might impact the interaction between these components. In the first aim of my work, I explored the ability of TIM-3 to bind membranes containing PS. I utilized surface plasmon resonance, an approach that mimics the lipid context TIM-3 might encounter in a cellular setting, to investigate the lipid binding of human TIM-3 and its relatives. I found that TIM-3 bound to PS-containing membranes with low micromolar affinity, weaker than its family members, but in range with other receptor-ligand pairs involved in regulating the immune response. I also demonstrated the importance of calcium and contacts with the hydrophobic PS acyl tail for TIM-3–PS binding. Using mutated variants of galectin-9, I showed that a potential role of this bivalent lectin is to crosslink TIM-3 to enhance its avidity for PS. In the second aim of my thesis, I asked how the TIM-3–PS interaction modulated T cell activity. In line with other reports in the literature, I found that TIM-3 promoted activity in the Jurkat T cell model across several readouts, including transcriptional activation and cytokine release. Interestingly, I observed that PS was exposed in Jurkat cell culture, which is not unexpected as cells in culture undergo apoptosis and activation, two processes in which PS can be exposed on the plasma membrane outer leaflet. Importantly, I demonstrated that the TIM-3 extracellular, ligand-binding region, and in particular, the ability to bind PS, contributed to TIM-3’s effect of promoting T cell activity in this model. Reducing PS binding with mutations or antibody blockade specifically reduced the ability of TIM-3 to promote T cell activity. Together, these findings support a role for PS as a ligand of TIM-3 that regulates the impact of this receptor on T cell signaling, thus enhancing our understanding of the regulation of TIM-3 by its ligand PS. Furthermore, they suggest that multiple ligands might simultaneously modulate TIM-3 to determine the nature of its effect. As therapies targeting TIM-3 move towards the clinic with the goal of enhancing anti-tumor immunity, it will be important to understand whether and how each ligand of TIM-3 modulates its activity in different immune cells to ensure optimal targeting of this unique immune-regulatory receptor.
Smith, Courtney McCaul, "Regulation of TIM-3 by Phosphatidylserine" (2021). Yale Graduate School of Arts and Sciences Dissertations. 415.