3D genome of CD8+ T cells reveals IRF8-mediated exhaustion in cancer [Hi-C]

CD8+ T cell responses are essential for anti-tumor immunity, but chronic antigen exposure in cancer can lead to T cell exhaustion, marked by high PD-1 expression. Recent studies have identified progenitor-like CD8+ T cells (Tprog cells) within tumor-infiltrating lymphocytes (TILs) that sustain antitumor responses. These cells can differentiate into terminally exhausted cells (Tterm cells), losing their proliferative and effector functions. Immunotherapy aims to enhance CD8+ TILs functionality, promoting their transition from Tprog to Tterm cells. However, the mechanisms behind this exhaustion remain unclear. Single-cell RNA sequencing and ATAC-seq have revealed distinct profiles and chromatin accessibility between progenitor and terminally exhausted states. Histone modifications predict a loss of enhancer-promoter contacts during this transition. Chromatin structure plays a crucial role in T cell differentiation. We constructed a high-resolution 3D genome map of CD8+ T cell subsets and, through multi-omics integration, identified chromatin structure changes linked to T cell exhaustion, including alterations in topologically associating domains (TADs) and chromatin loops, providing new insights into the genetic basis of CD8+ T cell exhaustion in cancer. Overall design: To investigate the role of CTCF in CD8? T cell chromatin organization, we performed CRISPR-Cas9-mediated Ctcf knockout in CD8? T cells isolated from Rosa26-Cas9 × OT-I transgenic mice. A single-guide RNA targeting Ctcf(forward: 5'-CACCGGATGAAGACTGAAGTCATGG; reverse: 5'-AAACCCATGACTTCAGTCTTCATCC) was cloned into a pWKO retroviral vector carrying an IRES-BFP cassette. Retrovirus was produced by co-transfecting the sgRNA vector and pCL-ECO into Phoenix cells using calcium phosphate, and supernatants were collected after 48 h. FACS-sorted naïve CD8? T cells were activated with anti-CD3/CD28 for 48 h and transduced with retrovirus via spinfection. Control (Empty, PB?) and Ctcf-knockout (FITC?) cells were mixed 1:1 and transferred into Tcrb?/? mice one day before E.G7 tumor implantation. On day 17, tumor-infiltrating exhausted CD8? T cells were sorted and analyzed by in situ Hi-C.