A genome-scale screen for synthetic drivers of T-cell proliferation

The engineering of patient T-cells for adoptive cell therapies has revolutionised the treatment of several cancer types. 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 functions and raise safety concerns due to permanent genome modification. Here we identify positive T-cell regulators via overexpression of ~12,000 barcoded human open reading frames (ORFs). The top-ranked genes increased primary human CD4+ and CD8+ T-cell proliferation, activation, and secretion of key cytokines like interleukin-2 and interferon-gamma. In addition, we developed a 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 beta receptor (LTBR), is typically expressed in myeloid cells but absent in lymphocytes. When overexpressed in T-cells, LTBR induced profound transcriptional and epigenomic remodelling, increasing T-cell effector functions, as well as resistance to exhaustion in chronic stimulation settings, via constitutive activation of the canonical NF-kappaB pathway. LTBR and other top-ranked genes improved antigen-specific chimeric antigen receptor (CAR) T-cell and gamma delta T-cell responses, highlighting their potential for future cancer-agnostic therapies5. Our results provide several strategies for improving next generation T-cell therapies via induction of new synthetic cell programmes. Overall design: Screen data: Primary T cells from three donors were transduced with a DNA barcoded ORF library. Best proliferating cells after restimulated were sorted and barcode abundance was compared to unsorted population. Single cell data: T cells overexpressing each ORF were separately transduced and cultured, and pooled only prior to the single cell experiment. Barcoded hashing antibodies were added for sample identification and overloading. Additionally, barcoded anti-NGFR antibody was added. In addition to transcriptome and TCR sequencing, direct ORF capture was performed. RNAseq: CD4 and CD8 T cells were transduced in triplicate with LTBR or tNGFR, and total RNA was extracted from resting or stimulated cells for 3' RNA sequencing in bulk. ATAC-seq: CD8 T cells were transduced in duplicate with LTBR or tNGFR; T cells were either rested or stimulated with CD3/CD28 for 24 h prior to nuclei isolation and tagmentation.