TIM-3 restrains anti-tumour immunity by regulating inflammasome activation.

Droplet-based massively parallel single-cell RNA sequencing (scRNAseq) was performed by encapsulating sorted live CD45+ tumour-infiltrating cells into droplets and libraries were prepared using Chromium Single Cell 5′ Reagent Kits v2 according to the manufacturer’s protocol (10X Genomics). The generated scRNA-seq libraries were sequenced using an Illumina HiSeq2500. Cells from each sample (tumour or dLN or ndLN from an individual mouse) was resuspended in PBS containing 2% FCS and stained with oligo tagged TotalSeq antibodies directed against CD45 for 30 min on ice. Cells were washed and pooled accordingly, centrifuged at 1,200 rcf for 5 min at 4 °C and resuspended in PBS + 2% FCS. For each 10X channel, 6 samples were combined: Havcr2fl/fl 1-Tumour, dLN, ndLN; KO1-Tumour, dLN, ndLN; Havcr2fl/fl 2-Tumour, dLN, ndLN; KO2- Tumour, dLN, ndLN.