By screening ~12,000 human ORFs overexpressed in primary human T cells, and using a novel method of single-cell sequencing coupled with ORF capture, Legut et al. identified genes spanning diverse functions that increased CD4+/CD8+ T cell activation via CD3/CD28. T cell expression of the top-ranked ORF, Lymphotoxin-β receptor, typically expressed by stromal and myeloid cells, induced NF-κB-dependent transcriptional and epigenomic changes to boost exhaustion/apoptosis resistance and proinflammatory cytokine secretion. Expression of top-ranked ORFs in CD19-CAR T cells and tumor-reactive γδ T cells improved their antigen-specific responses.
Contributed by Paula Hochman
ABSTRACT: The engineering of autologous patient T cells for adoptive cell therapies has revolutionized the treatment of several types of cancer1. However, further improvements are needed to increase response and cure rates. CRISPR-based loss-of-function screens have been limited to negative regulators of T cell functions2-4 and raise safety concerns owing to the permanent modification of the genome. Here we identify positive regulators of T cell functions through overexpression of around 12,000 barcoded human open reading frames (ORFs). The top-ranked genes increased the proliferation and activation of primary human CD4+ and CD8+ T cells and their secretion of key cytokines such as interleukin-2 and interferon-γ. In addition, we developed the single-cell genomics method OverCITE-seq for high-throughput quantification of the transcriptome and surface antigens in ORF-engineered T cells. The top-ranked ORF-lymphotoxin-β receptor (LTBR)-is typically expressed in myeloid cells but absent in lymphocytes. When overexpressed in T cells, LTBR induced profound transcriptional and epigenomic remodelling, leading to increased T cell effector functions and resistance to exhaustion in chronic stimulation settings through constitutive activation of the canonical NF-κB pathway. LTBR and other highly ranked genes improved the antigen-specific responses of chimeric antigen receptor T cells and γδ T cells, highlighting their potential for future cancer-agnostic therapies5. Our results provide several strategies for improving next-generation T cell therapies by the induction of synthetic cell programmes.