Galectin-9 interacts with PD-1 and TIM-3 to regulate T cell death and is a target for cancer immunotherapy

The two T cell inhibitory receptors PD-1 and TIM-3 are co-expressed during exhausted T cell differentiation, and recent evidence suggests that their crosstalk regulates T cell exhaustion and immunotherapy efficacy; however, the molecular mechanism is unclear. Here we show that PD-1 contributes to the persistence of PD-1+TIM-3+ T cells by binding to the TIM-3 ligand galectin-9 (Gal-9) and attenuates Gal-9/TIM-3-induced cell death. Anti-Gal-9 therapy selectively expands intratumoral TIM-3+ cytotoxic CD8 T cells and immunosuppressive regulatory T cells (Treg cells). The combination of anti-Gal-9 and an agonistic antibody to the co-stimulatory receptor GITR (glucocorticoid-induced tumor necrosis factor receptor-related protein) that depletes Treg cells induces synergistic antitumor activity. Gal-9 expression and secretion are promoted by interferon β and γ, and high Gal-9 expression correlates with poor prognosis in multiple human cancers. Our work uncovers a function for PD-1 in exhausted T cell survival and suggests Gal-9 as a promising target for immunotherapy.

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Flow cytometry data were analyzed using FlowJo 10.6.2 (BD). Western blotting and Duolink data were processed and analyzed using ImageJ 2.0.0-rc-69/1.52p (NIH). Statistical tests were run using Prism 8.4.1 (GraphPad). CyTOF data were analyzed using Cytobank (Cytobank Inc.). Single-cell RNA-seq data of human melanoma TILs (GSE120575) were reanalyzed with BBrowser2 (BioTuring). The heatmaps that show the expression of the indicated markers were generated using pheatmap R package version 1.0.12.

October 2018
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Age and sex-matched animals were used for each experiment. Mice were randomized prior to tumor size measurement and antibody treatment. For experiments other than mice studies, samples were also randomly allocated into experimental groups.
Blinding was not performed in mouse experiments because investigator needed to know the treatment groups in order to perform the study. Bias are effectively alleviated as for both in vivo and in vitro studies, the robust phenotype of our results is based on objective measurements instead of any human estimation.
CyTOF antibodies are listed in supplementary table 1. All other antibodies used in the study are described in supplemental table 2.
All antibodies used are commercially available and validated by the manufacturers.