Single-cell transcriptomics has transformed our ability to characterize cell states, but deep biological understanding requires more than a taxonomic listing of clusters. As new methods arise to ...measure distinct cellular modalities, a key analytical challenge is to integrate these datasets to better understand cellular identity and function. Here, we develop a strategy to “anchor” diverse datasets together, enabling us to integrate single-cell measurements not only across scRNA-seq technologies, but also across different modalities. After demonstrating improvement over existing methods for integrating scRNA-seq data, we anchor scRNA-seq experiments with scATAC-seq to explore chromatin differences in closely related interneuron subsets and project protein expression measurements onto a bone marrow atlas to characterize lymphocyte populations. Lastly, we harmonize in situ gene expression and scRNA-seq datasets, allowing transcriptome-wide imputation of spatial gene expression patterns. Our work presents a strategy for the assembly of harmonized references and transfer of information across datasets.
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•Seurat v3 identifies correspondences between cells in different experiments•These “anchors” can be used to harmonize datasets into a single reference•Reference labels and data can be projected onto query datasets•Extends beyond RNA-seq to single-cell protein, chromatin, and spatial data
A computational approach to integrate diverse modalities associated with single-cell sequencing datasets can be used to better understand cellular identity and function.
SARS-CoV-2 infection and vaccination elicit potent immune responses. Our study presents a comprehensive multimodal single-cell analysis of blood from COVID-19 patients and healthy volunteers ...receiving the SARS-CoV-2 vaccine and booster. We profiled immune responses via transcriptional analysis and lymphocyte repertoire reconstruction. COVID-19 patients displayed an enhanced interferon signature and cytotoxic gene upregulation, absent in vaccine recipients. B and T cell repertoire analysis revealed clonal expansion among effector cells in COVID-19 patients and memory cells in vaccine recipients. Furthermore, while clonal αβ T cell responses were observed in both COVID-19 patients and vaccine recipients, expansion of clonal γδ T cells was found only in infected individuals. Our dataset enables side-by-side comparison of immune responses to infection versus vaccination, including clonal B and T cell responses. Our comparative analysis shows that vaccination induces a robust, durable clonal B and T cell responses, without the severe inflammation associated with infection.
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•Both COVID-19 infection and immunization elicited robust adaptive immune responses•SARS-CoV-2 infection results in profound upregulation of type I IFN signaling•Immune cells of COVID-19 patients have elevated expression of cytotoxic genes•Both infection and vaccine lead to clonal expansion of T cells and SHM in B cells
Immunology; Immune response; Transcriptomics
High-throughput single-cell RNA sequencing has transformed our understanding of complex cell populations, but it does not provide phenotypic information such as cell-surface protein levels. Here, we ...describe cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq), a method in which oligonucleotide-labeled antibodies are used to integrate cellular protein and transcriptome measurements into an efficient, single-cell readout. CITE-seq is compatible with existing single-cell sequencing approaches and scales readily with throughput increases.
Adenoid cystic carcinoma (ACC) is a rare cancer type that originates in the salivary glands. Tumors commonly invade along nerve tracks in the head and neck, making surgery challenging. Follow-up ...treatments for recurrence or metastasis including chemotherapy and targeted therapies have shown limited efficacy, emphasizing the need for new therapies. Here, we report a Drosophila-based therapeutic approach for a patient with advanced ACC disease. A patient-specific Drosophila transgenic line was developed to model the five major variants associated with the patient's disease. Robotics-based screening identified a three-drug cocktail—vorinostat, pindolol, tofacitinib—that rescued transgene-mediated lethality in the Drosophila patient-specific line. Patient treatment led to a sustained stabilization and a partial metabolic response of 12 months. Subsequent resistance was associated with new genomic amplifications and deletions. Given the lack of options for patients with ACC, our data suggest that this approach may prove useful for identifying novel therapeutic candidates.
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•Personalized therapy was developed for patient with Adenoid Cystic Carcinoma•Genomics analysis was leveraged to establish a Drosophila ‘personalized patient avatar’•A robotics-based screen identified a novel three drug therapeutic cocktail•12 months response was followed by relapse and significant tumor genomic re-wiring
Molecular physiology; genetics; biotechnology; cancer systems biology
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. 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 therapies
. Our results provide several strategies for improving next-generation T cell therapies by the induction of synthetic cell programmes.
Defining the transcriptomic identity of malignant cells is challenging in the absence of surface markers that distinguish cancer clones from one another, or from admixed non-neoplastic cells. To ...address this challenge, here we developed Genotyping of Transcriptomes (GoT), a method to integrate genotyping with high-throughput droplet-based single-cell RNA sequencing. We apply GoT to profile 38,290 CD34
cells from patients with CALR-mutated myeloproliferative neoplasms to study how somatic mutations corrupt the complex process of human haematopoiesis. High-resolution mapping of malignant versus normal haematopoietic progenitors revealed an increasing fitness advantage with myeloid differentiation of cells with mutated CALR. We identified the unfolded protein response as a predominant outcome of CALR mutations, with a considerable dependency on cell identity, as well as upregulation of the NF-κB pathway specifically in uncommitted stem cells. We further extended the GoT toolkit to genotype multiple targets and loci that are distant from transcript ends. Together, these findings reveal that the transcriptional output of somatic mutations in myeloproliferative neoplasms is dependent on the native cell identity.
The expression of inhibitory immune checkpoint molecules, such as programmed death-ligand (PD-L)1, is frequently observed in human cancers and can lead to the suppression of T cell-mediated immune ...responses. Here, we apply expanded CRISPR-compatible (EC)CITE-seq, a technology that combines pooled CRISPR screens with single-cell mRNA and surface protein measurements, to explore the molecular networks that regulate PD-L1 expression. We also develop a computational framework, mixscape, that substantially improves the signal-to-noise ratio in single-cell perturbation screens by identifying and removing confounding sources of variation. Applying these tools, we identify and validate regulators of PD-L1 and leverage our multimodal data to identify both transcriptional and post-transcriptional modes of regulation. Specifically, we discover that the Kelch-like protein KEAP1 and the transcriptional activator NRF2 mediate the upregulation of PD-L1 after interferon (IFN)-γ stimulation. Our results identify a new mechanism for the regulation of immune checkpoints and present a powerful analytical framework for the analysis of multimodal single-cell perturbation screens.
Most variants associated with complex traits and diseases identified by genome-wide association studies (GWAS) map to noncoding regions of the genome with unknown effects. Using ancestrally diverse, ...biobank-scale GWAS data, massively parallel CRISPR screens, and single-cell transcriptomic and proteomic sequencing, we discovered 124
-target genes of 91 noncoding blood trait GWAS loci. Using precise variant insertion through base editing, we connected specific variants with gene expression changes. We also identified
-effect networks of noncoding loci when
target genes encoded transcription factors or microRNAs. Networks were themselves enriched for GWAS variants and demonstrated polygenic contributions to complex traits. This platform enables massively parallel characterization of the target genes and mechanisms of human noncoding variants in both
and
.
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