Altered costimulatory signaling and hypoxia support chromatin landscapes that limit the functional potential of exhausted T cells in cancer [RNA-seq]

Increases in terminally exhausted T cells in the tumor are associated with poor responses to immunotherapy, yet the mechanisms that promote progression to terminal exhaustion remain undefined. To understand the effect of epigenetic changes in subsets of tumor-infiltrating CD8+ T cells, we performed RNA-seq to understand changes in gene expression. CD8 T cells were sorted from the murine B16 melanoma tumor using PD1 and Tim3 expression to define four subsets: PD1lo, PD1mid, PD1hi, and PD1hiTim3+. Additional control samples include paired CD44+ cells from the tumor-draining lymph node of tumor bearing mice, and OT-I effector CD8 T cells isolated from Vaccinia-ova infection. CD8 TIL PD1hi Tim3+ from B16 tumors treated with IgG control, anti-PD1 or anti-41BB agonist immunotherapies were also isolated for RNAseq. Overall design: Three individual biological replicates of each sorted population: dLN CD8+ CD44+; TIL CD8+ PD1lo; TIL CD8+ PD1mid; TIL CD8+ PD1hi; TIL CD8+ PD1hi Tim3+; OT-I CD8+ effector from d10 Vaccinia-Ova infection; TIL CD8+ PD1hi Tim3+ IgG control treated; TIL CD8+ PD1hi Tim3+ anti-PD1 treated; TIL CD8+ PD1hi Tim3+ anti-4-1BB treated