Altered costimulatory signaling and hypoxia support chromatin landscapes that limit the functional potential of exhausted T cells in cancer [CUT&RUN]

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. We profiled the chromatin landscape of subsets of tumor-infiltrating CD8+ T cells using CUT&RUN. 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. We performed CUT&RUN for H3K4me3, H3K27me3, H3K27ac, and H3K9ac, as well as the transcription factors Tox and Batf. Overall design: Two 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) per histone modification mark (H3K4me3; H3K27me3; H3K27ac; H3K9ac) or transcription factor (Tox) profiled, except Batf which has one replicate.