TLE transcription cofactors in anti-viral and anti-tumor immunity

CD8+ T lymphocytes are essential players in mounting protective cellular immune responses against pathogens and malignantly transformed cells. However, exposure to persisting antigens due to chronic viral infections or tumor microenvironment drives CD8+ T cells into an exhausted/hypofunctional state, limiting the efficacy of immunotherapy. There is an urgent need to molecularly reconstruct the exhaustion process and identify actionable therapeutic targets to alleviate exhaustion and restore durable functions in T cells. Transcriptional and epigenetic regulation has essential roles in initiation, fate decision, differentiation, and stability of the exhausted CD8+ T (Tex) cells. Transcription cofactors do not bind DNA directly, but have the unique ability of interacting with multiple sequence-specific transcription factors (TFs) and form integrative regulatory complexes. Our preliminary studies on Tle3 cofactor revealed its distinct impact on Tex cells elicited by chronic viral infection. We hypothesize that Tle3 forges a critical linker that integrates TFs and epigenetic regulators and controls Tex functions via distinct regulatory complexes. Ectopic expression of an engineered human TLE3 variant that lacked the C-terminal WDR domain (called TLE3-DWDR) in hCD19- CAR-T cells enhanced their anti-leukemia capacity in a xenograft model. We further hypothesize that molecular engineering of Tle3 decouples its 'preferred' regulatory effects from the 'unwanted' ones and can be leveraged to enhance CAR-T cells in tumor control. We will test these through two aims: Aim 1, to dissect Tle3-mediated complex assembly in directing Tex cell fate and persistence. We will use target gene-specific and genome-wide approaches to dissect the assembly of Tle3- nucleated complexes, including 1) Tle3 recruitment by TFs mobilized by persistent antigen stimulation, 2) TF- Tle3 cooperativity in engaging Mll4 to deploy H3K4me1/2 for enhancer initialization, 3) TF-Tle3 cooperativity in engaging Hdac1 and/or Ezh2 for enhancer inactivation, and 4) essential requirements for Tle3 in stabilizing the TF-epigenetic regulator complexes. Aim 2, to expand the reprogramming capacity by engineered human TLE3 to enhance CAR-T cells. We will pursue the following goals: 1) to test if the beneficial effects of TLE3-DWDR are broadly applicable to different CAR binders, resulting in improved controls over leukemia relapse and solid tumors, 2) to investigate the mechanisms underlying TLE3-DWDR-mediated reprogramming: reshaping existing TLE3- nucleated complexes and/or acquiring de novo complexes, and 3) to refine TLE3 engineering via domain and contact surface optimization to boost CAR-T effector functions. These efforts will not only improve the understanding of T cell exhaustion process with increased precision and resolution, but also establish TLE3 functional decoupling as an actionable strategy to avert T cell exhaustion and achieve durable immune control over chronic infections and cancers.