RNA-sequencing of PD-1+ CD8 T cells following two week PD-1 blockade (stem-like and terminally-differentiated subsets).

The goal of this study is to characterize the transcriptional profile of PD-1+ stem-like (Tim-3-CD73+) versus terminally-differentiated (Tim-3+CD73-) CD8 T cells following two week aPD-1 blockade. Overall design: Splenocytes were isolated from mice >45 days post-LCMV Clone 13 infection (2×106 pfu, i.v. after CD4 T cell depletion). Mice received isotype or aPD-1 blockade for two weeks. Cells were stained with extracellular antibodies (CD4, CD8, CD19, PD-1, Tim-3, and CD73 from Biolegend) for 30 minutes. Cell viability was determined with the Live/Dead fixable aqua dead cell stain kit (Invitrogen). Cells were washed and sorted on a FACSAria II (BD Biosciences). Two populations of PD-1+ CD8 T cells were sorted in duplicate from aPD-1 or isotype-treated samples: stem-like (Tim-3-CD73+) and terminally-differentiated (Tim-3+CD73-). CD44loCD62Lhi CD8 T cells were also sorted in duplicate from naïve mice as controls. RNA was isolated from each sample using the All-Prep DNA/RNA Micro Kit (Qiagen) and then submitted for sequencing.