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

Fig. 3

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.

Mateusz Legut, Zoran Gajic, Maria Guarino, Daniloski Z, Rahman JA, Xue X, Lu C, Lu L, Mimitou EP, Hao S, Davoli T, Diefenbach C, Smibert P, Sanjana NE. (2022) A genome-scale screen for synthetic drivers of T cell proliferation. Nature [Epub ahead of print]. [abstract]

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