Mechanism of TIM-3 Inhibition in Th1 Cells

Abstract

TIM-3 is a co-inhibitory receptor that regulates Interferon(IFN)-g-secreting CD4+ T helper 1 (Th1) cells and marks CD8+ T cells that are terminally exhausted. Considering that anti-TIM-3 antibodies are being investigated in clinical trials for different tumors, it is important to understand the mechanism of its inhibitory function in CD8+ cytotoxic T cells and CD4+ Th1 cells. However, TIM-3 lacks a canonical cytoplasmic inhibitory signaling motif. Bat3 has been shown to be associated with TIM-3 and its absence promotes a dysfunctional state in Th1 cells. Cbl-b, an E3 ligase, whose inhibitory function in T cell signaling is well defined, was also found to bind TIM-3. We therefore hypothesized a model in which Bat3 binding to TIM-3 allows a permissive state of T cell function, whereas upon TIM-3 stimulation, the release of Bat3 and binding of Cbl-b results in an inhibitory state. In this study, we show that the expression of Cbl-b and Bat3 are correlated with that of TIM-3 in in vitro cultured Th1 cells and in CD8+ tumor-infiltrating lymphocytes (TILs) from B16-OVA tumors. We confirmed that Cbl-b associates with TIM-3 by co-immunoprecipitation, which is abrogated when Cbl-b is locked in a closed conformation. We found that Cbl-b binds to the same region on the cytoplasmic tail of TIM-3 as Bat3. Unlike Bat3 where galectin-9-mediated phosphorylation of TIM-3 triggers dissociation, phosphorylation increases Cbl-b binding, suggesting that phosphorylation may partially explain the switch from Bat3 binding to Cbl-b binding. We also found that the loss of Bat3 increases Cbl-b binding to TIM-3 to an extent beyond that driven by phosphorylation alone, indicating competition between Bat3 and Cbl-b for binding to TIM-3. This study is the first to investigate the interplay between TIM-3, Bat3, and Cbl-b for the purpose of elucidating the mechanism by which TIM-3 signaling inhibits T cell function, promoting the understanding of T cell exhaustion and anti-tumor immunity.