G2 - Immune Targeting and Approaches with Genetically-Modified Cells and Cell Therapies (Including CAR-T, CAR-NK, TCR editing)
185: Discovering Novel Transcriptional Regulators of T Cell Exhaustion for Epigenetic Reversal of T Cell Dysfunction
Type: Oral Abstract Session
Presentation Details
Session Title: CAR T-cell Therapies
T cells are part of the immune system’s adaptive defense; they specifically target and kill both virally infected and cancerous cells through antigen recognition. Unfortunately, cancer can exploit intrinsic T cell mechanisms to survive attack by the immune system. T cell exhaustion arises from chronic antigen stimulation, which shifts a portion of the T cell population to the exhausted state (TEX), resulting in diminished T cell proliferation and tumor/viral clearance. Modern genome engineering technologies have the potential to dramatically advance T cell therapy by programming TEX cells into desirable phenotypic states. The transcription factor (TF) TOX drives and maintains the TEX state through epigenetic regulation of exhaustion-associated programs. To recapitulate T cell exhaustion in vitro, we repeatedly stimulated primary human T cells with anti-CD3/CD28 beads to mimic chronic stimulation and confirmed induction of the exhaustion program via RNA- and ATAC-seq. Specifically, multiple genomic regions within the TOX locus became differentially accessible with in vitro chronic stimulation. Through a CRISPR interference (CRISPRi) screen tiling the TOX locus, we confirmed modulation of TOX expression by targeting these accessible non-coding regions, emphasizing the highly regulated epigenetic control of TOX downstream of chronic stimulation. To understand what other TF regulators might be driving or preventing T cell exhaustion upstream of TOX, we designed two screens using a CRISPR activation (CRISPRa) approach targeting all genes encoding TFs in the human genome or an Open Reading Frame (ORF) library encoding all the TFs in the human genome with TOX protein expression as a readout. CD8+ T cells from three distinct donors were used in an acute stimulation setting (one TCR stimulation with anti-CD3/CD28 beads) and a chronic stimulation setting (two additional stimulations). Comparing screen results in the context of acute versus chronic stimulation facilitated identification of factors specific to the TEX state. The ORF screen yielded more TF hits that were modulating TOX expression than the CRISPRa screen (3 gRNA hits versus >100 ORF hits). Over 100 TF ORFs were enriched in TOX-high and TOX-low bins, including both known and novel regulators of TOX. Individual validations of top enriched TFs unique to chronic stimulation confirmed modulation of TOX expression in the expected direction at the protein and transcript level. FACS staining of exhaustion (LAG3, PD-1, and TIGIT) and memory (IL7R) surface markers revealed significant differences in expression levels, suggesting potential phenotypic changes in T cell function. Ongoing work is focused on validating the effects of these TFs on T cell function (T cell killing of cancer cells and cytokine expression assays), transcriptome (RNA-seq), and epigenome (ATAC-seq). In addition, we are testing these candidate TFs to reprogram tumor-infiltrating lymphocytes from primary lung tumor resections. Through epigenetic reprogramming, we hope to achieve clinically relevant T cell phenotypes to improve current immunotherapies in both viral infection and cancer contexts.
Christian McRoberts Amador1, Aretha Gao2, Sean McCutcheon3, Charles A. Gersbach3
1Pharmacology and Cancer Biology, Duke University, Durham, NC,2Biology, Duke University, Durham, NC,3Biomedical Engineering, Duke University, Durham, NC"
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