Chronic Type I interferons signaling promotes lipid peroxidation-driven terminal CD8+T cell exhaustion and curtails anti-PD-1 treatment efficacy [ATAC-Seq]

Identifying signals that govern the differentiation of tumor-infiltrating CD8 + T cells (CD8 + TILs) towards exhaustion can improve current therapeutic approaches for cancer. Here, we show that type I interferons (IFN-Is) act as environmental cues enhancing terminal CD8 + T cell exhaustion in tumors. We found enrichment of IFNIs-stimulated genes (ISGs) within exhausted CD8 + T cells (Tex cells) in patients across various cancer types, with heightened ISG levels correlating with poor response to immune checkpoint blockade (ICB) therapy. In preclinical models, CD8 + TILs devoid of IFN-Is signaling developed less exhaustion features, provided better tumor control and showed greater response to ICB-mediated rejuvenation. Mechanistically, chronic IFN-Is stimulation perturbed lipid metabolism and redox balance in Tex cells, leading to aberrant lipid accumulation and elevated oxidative stress. Collectively, these defects promoted lipid peroxidation, which potentiated metabolic and functional exhaustion of Tex cells. Thus, cell intrinsic IFN-Is signaling regulates the extent of CD8+ TIL exhaustion, and has important implications for immunotherapy. CD8+T cells were isolated using Human CD8+ T Cell Isolation Kit (Miltenyi Biotec) according to the manufacturers procedure. Purified CD8+T cells were then stimulated with anti-CD3/anti-CD28-coated microbeads (Dynabeads M-450 Epoxy, Invitrogen; bead to cell ratio of 1:1) in the presence of 10U/ml recombinant human IL-2 (PeproTech) in T cell medium (RPMI 1640 medium supplemented with 10% FBS, 50M -mercapteoethanol, 1M HEPES, 1X non-essential amino acids (NEAA), 100mM sodium pyruvate, and 1% penicillin-streptomycin). Surviving cells were passaged every 48 hours with fresh microbeads and IL-2 until day2 or day 8 to generate Teff or Tex cells. In these conditions, human universal Type I interferon (PBL Assay science) was added at 1000U/mL throughout the culture. 50,000 alive CD8+ T cells were sorted through FACs with Influx (BD Biosciences) for establishing ATAC-seq library.