Single-cell RNA sequencing of T3 sarcoma tumors following treatment with CD8-mIL2, CTRL-not-alpha-mIL2, or anti-PD-1

To assess treatment-induced changes of intratumoral CD8+ T cells in an unbiased manner, CD8+ TILs from T3 sarcomas were analyzed using single-cell RNA-sequencing (scRNAseq) 2 or 4 days after therapy with CD8-mIL2, CTRL-not-α-mIL2, anti-PD-1, or combinations thereof. Barcoded peptide:MHC reagents were included to identify mLama4-specific CD8+ T cells. CD8-mIL2 therapy promotes the generation of a new stem-like effector CD8+ T cell cluster with a signature indicative of polyfunctionality and with features of T cell memory. T3 tumor-bearing mice were treated on day twelve post-tumor transplant. Two and four days later, single-cell suspensions were prepared from TILs. Total CD8+ T cells were enriched using a CD8+ T cell positive selection kit (Miltenyi). Tumors from individual mice were labeled with one of the following antibodies before pooling in order to identify cells from individual tumors in downstream analysis: 1) TCR (TotalSeq™-C0120 anti-mouse TCR β chain antibody, Cat# 109259, BioLegend), 2) CD90.2 (TotalSeq™-C0075 anti-mouse CD90.2 (Thy1.2) antibody, Cat#, 105353, BioLegend), and 3) CD45 (TotalSeq™-C0096 anti-mouse CD45 antibody, Cat# 103169, BioLegend). Samples were submitted to the Genome Institute at Washington University in St. Louis to generate 10x libraries using 10x 5'v2 Single Cell RNAseq, V(D)J enrichment, feature barcoding, and Barcode Enabled Antigen Mapping kits. Fastq files were processed using the Cell Ranger (v7.1.0) multi pipeline with the default parameters. Gene expression libraries were aligned to the mouse transcriptome (mm10-2020-A) and TCR sequences were aligned to the VDJ reference dataset (GRCm38-alts-ensembl-5.0.0).