Orthogonal screens to decode human T cell state and function

In the last decade, the paradigm for cancer therapy has incrementally transitioned away from non-specific cytotoxic therapies (radiation, chemotherapy) and targeted therapies (small molecules, biologics) and towards immune cell-based therapies. Immune cell-based therapies such as adoptive T cell therapy (ACT) harness the intrinsic ‘sense and respond’ functions of immune cells to selectively target and eliminate cancer cells. Nevertheless, more than half of cancer patients either do not respond or relapse to existing ACTs. Several studies have defined specific transcriptional and epigenetic signatures of the infused T cell product associated with clinical response, indicating that T cell state and fitness is linked to ACT efficacy. Thus, epigenetically reprogramming T cells with enhanced potency and durability has the potential to improve ACT. However, this potential has yet to be fully realized due to technical challenges of adapting CRISPR-based epigenome editing technologies for applications in primary human T cells. To overcome these challenges, we developed and rigorously characterized compact and robust CRISPR repressors and activators for endogenous gene regulation. Next, we leveraged these technologies to systematically interrogate the effects of >100 transcriptional and epigenetic regulators on human CD8+ T cell state and function through complementary CRISPR interference (CRISPRi) and activation (CRISPRa) screens. These CRISPRi/a screens converged on basic leucine zipper ATF-like transcription factor (BATF3). Subsequent assays revealed that BATF3 overexpression promotes specific features of memory T cells (such as increased expression of IL7R and glycolysis), counters T cell exhaustion, and enhances CAR T cell potency in both in vitro and in vivo tumor models. In addition, BATF3 programs a transcriptional profile strongly associated with positive clinical response to CD19 CAR T cell therapy. Given that BATF3 is a compact transcription factor (TF) without any transactivation or epigenetic domains, we speculated that BATF3 achieves its widespread effects by interacting with other TFs. To identify these factors, we conducted parallel CRISPR knockout screens targeting all TFs with or without BATF3 overexpression. Using IL7R expression as a proxy for BATF3 activity, we identified both BATF3-independent and dependent transcriptional regulators of IL7R expression. For example, JUNB and IRF4 were uniquely enriched in the low IL7R population in the screen with BATF3 overexpression, suggesting BATF3 heterodimerizes with JUNB and interacts with IRF4 to regulate gene expression. Finally, these CRISPR knockout screens illuminated other candidate therapeutic targets for future exploration and characterization. Overall, we have developed a widely applicable synthetic biology toolkit of orthogonal epigenome editors, which we used to systematically identify regulators of human CD8+ T cell state and function. This catalogue of regulators could serve as the basis for engineering next generation T cell therapies for cancer.