TIGIT contributes to the regulation of 4-1BB and does not define NK cell dysfunction in glioblastoma

This study evaluates the genotype of NK cells sorted for expression of TIGIT into TIGIThigh, TIGITmedium and TIGITnegative NK cells. RNAseq was performed on these three NK cell populations. Human NK cells isolated from PBMCs were sorted as TIGIThigh, TIGITmedium and TIGITnegative, and total RNA was isolated using the mirVana miRNA Isolation Kit (Thermo Fisher Scientific, Waltham, MA) according to manufacturer instructions, with >700 ng of RNA per sample. All samples were checked for quality prior to analysis by Agilent Bioanalyzer, with DV200 > 80 and RIN > 8.4 for all samples. Samples were sent to GeneWiz for library preparation and sequencing. Each sample contained at least 30 million paired-end reads with length of 250 bp. Data quality control (adapter trimming, minimum Phred score 30, minimum read length 50 bp) was performed using fastp 27 (version 0.19.5). Quality trimmed reads were mapped to human reference genome (GRCh38) using the STAR aligner 28 (version 2.7.9a). Reads aligned to each gene feature were counted using the featureCounts 29 program from Subread package (version 1.6.1). Differential expression (DE) analysis was performed using the edgeR method 30 (version 3.32.1) and genes with False Discovery Rate (FDR) ≤ 0.01 were denoted as significant.