OverCITE-seq – high-throughput quantification of the transcriptome and surface antigens in ORF-engineered T cells.

The engineering of autologous patient T cells for adoptive cell therapies has revolutionized the treatment of several types of cancer. 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 owing to the permanent modification of the genome.

Researchers from the New York Genome Center have identified 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, the researchers 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 therapies. These results provide several strategies for improving next-generation T cell therapies by the induction of synthetic cell programmes.

Single-cell OverCITE-seq identifies shared and distinct transcriptional programs
that are induced by gene overexpression in T cells

a, OverCITE-seq captures overexpression (ORF) constructs, transcriptomes, TCR clonotypes, cell-surface proteins and treatment hashtags in single cells. b, ORF assignment rate in resting and CD3/CD28-stimulated T cells. c, Antibody-derived tag sequencing (ADTs; right) yields similar NGFR expression in tNGFR-transduced T cells to flow cytometry (left) with tNGFR-transduced T cells. Untransduced cells (left) or cells assigned a non-tNGFR ORF (right) are shown in grey. d, Uniform manifold approximation and projection (UMAP) representation of single-cell transcriptomes after unsupervised clustering of OverCITE-seq-captured ORF singlets. The inset in the top left identifies stimulated and resting T cells as given by treatment hashtags. For each cluster, a subset of the top 20 differentially expressed genes is shown. HIST1H1B is also known as H1-5 and HIST1H3C is also known as H3C3. e, ORF prevalence in two representative clusters. Standardized residual values are from a chi-squared test. ORFs of interest are shown.