Background Tim-3, or transmembrane immunoglobulin and mucin domain-3, is a type I membrane protein expressed by various immune cell types and which has been shown to have a co-stimulatory role in T cells through the PI3K pathway.^{1 2} Comprising an extracellular, a transmembrane and an intracellular domain containing five tyrosine residues, we and others have shown that Tim-3 participates in TCR-mediated signaling pathways. Using LCMV infection, we also found that Tim-3 expression influences the formation of short-lived effector and memory precursor CD8⁺ T cells. We hypothesize that Tim-3 enhances CD8⁺ T cell function in acute infections and contributes to enhanced CD8⁺ effector function.

Methods To test this hypothesis, we have used LCMV-specific TCR transgenic (P14) mice expressing a truncated version of Tim-3 and CD8⁺ conditional deletion of Tim-3. CD8⁺ T cell activation and memory were analyzed by flow cytometry after acute infection with LCMV Armstrong. Bulk RNA and TCR sequencing was performed on endogenous Tim-3 expressing and non expressing effector CD8+ cells. Nuclear localization of Foxo1 was analyzed using Amnis Imagestream.

Results At the effector stage, Tim-3⁺ T cells had increased expression of effector CD8 markers such as T-bet. Tim-3 knockout mice also showed a significantly lower number of LCMV-specific T cells. Additionally, endogenous Tim-3⁺ T cells were also significantly better at cytokine production and had increased cytotoxicity. Tim-3 expression correlated with better cell survival in homeostatic conditions and during reactivation induced cell death. Through bulk RNA and TCR sequencing, we have found that Tim-3⁺ effector CD8⁺ T cells also have a distinct pattern of upregulation of effector genes and show increased expansion of TCR clones. Mechanistically, we also show that Tim-3 signals via Foxo1, inhibiting its entry into the cytoplasm, thus conferring an enhanced effector function.

Conclusions Our data suggest that Tim-3 signaling positively contributes to an effective CD8⁺ T cell response in an acute infection. These findings may lead to a better understanding of CD8⁺ T cell function in different settings, including in the tumor microenvironment.

Acknowledgements We would like to thank Angela Gocher-Demske for titration of LCMV Armstrong and Alex Rowe for assistance with Xcelligence cytotoxicity assays.

References

1. Ferris RL, Lu B, Kane LP. Too much of a good thing? Tim-3 and signaling in T cell exhaustion. J Immunol. 2014;193:1525–1530

2. Avery L, Filderman J, Szymczak-Workman AL, Kane LP. Tim-3 co-stimulation promotes short-lived effector T cells, restricts memory precursors, and is dispensable for T cell exhaustion. Proc Natl Acad Sci USA. 2018;115:2455–2460

Ethics Approval All mouse experiments were performed in accordance with protocols approved by the University of Pittsburgh Institutional Animal Care and Use Committee