Multiplexed, single-cell profiling of histone modifications with SCEPTRE v1 (protocols.io.buaynsfw)

AbstractThe development of the mouse central nervous system (CNS) involves coordinated execution of transcriptional and epigenetic programs. These programs have been extensively studied through single-cell technologies in a pursuit to characterize the underlying cell heterogeneity. However, histone modifications pose additional layers of both positive and negative regulation that defines cellular identity. Here we show that the Cut&Tag technology can be coupled with a droplet-based single cell library preparation platform to produce high quality chromatin modifications data at a single cell resolution in tens of thousands of cells. We apply single-cell Cut&Tag (scC&T) to probe histone modifications characteristic of active promoters (H3K4me3), active promoters and enhancers (H3K27ac), active gene bodies (H3K36me3) and inactive regions (H3K27me3) and generate scC&T profiles for almost 50,000 cells. scC&T profiles of each of these histone modifications were sufficient to determine cell identity and deconvolute at single cell level regulatory principles such as promoter bivalency, spreading of H3K4me3 and promoter-enhancer connectivity. Moreover, we used scC&T to investigate the single-cell chromatin occupancy of transcription factor Olig2 and the cohesin complex component Rad21. Our results indicate that analysis of histone modifications and transcription factor occupancy at a single cell resolution can provide unique insights of epigenomic landscapes in the CNS. We also provide an online resource that can be used to interactively explore the data at https://castelobranco.shinyapps.io/BrainCutAndTag2020/.

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Faculty Opinions recommendation of Single cell profiling of potentiated phospho-protein networks in cancer cells.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1020412.323536 ◽

2005 ◽

Author(s):

Alfonso Martinez Arias

Keyword(s):

Single Cell ◽

Cancer Cells ◽

Protein Networks ◽

Single Cell Profiling

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Faculty Opinions recommendation of Single cell profiling of potentiated phospho-protein networks in cancer cells.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1020412.232570 ◽

2004 ◽

Author(s):

James Woodgett

Keyword(s):

Single Cell ◽

Cancer Cells ◽

Protein Networks ◽

Single Cell Profiling

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Faculty Opinions recommendation of Single-cell profiling of the developing mouse brain and spinal cord with split-pool barcoding.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.732844079.793548900 ◽

2018 ◽

Author(s):

Yann Herault

Keyword(s):

Spinal Cord ◽

Single Cell ◽

Mouse Brain ◽

Single Cell Profiling ◽

Developing Mouse Brain ◽

Brain And Spinal Cord

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Faculty Opinions recommendation of Simultaneous single-cell profiling of lineages and cell types in the vertebrate brain.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.732909626.793544340 ◽

2018 ◽

Author(s):

Bruce Appel

Keyword(s):

Single Cell ◽

Cell Types ◽

Vertebrate Brain ◽

Single Cell Profiling

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Single-cell profiling of myeloid cells in glioblastoma across species and disease stage reveals macrophage competition and specialization

Nature Neuroscience ◽

10.1038/s41593-020-00789-y ◽

2021 ◽

Author(s):

Ana Rita Pombo Antunes ◽

Isabelle Scheyltjens ◽

Francesca Lodi ◽

Julie Messiaen ◽

Asier Antoranz ◽

...

Keyword(s):

Single Cell ◽

Myeloid Cells ◽

Disease Stage ◽

Single Cell Profiling

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Designed improvement to T-cell immunotherapy by multidimensional single cell profiling

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-001877 ◽

2021 ◽

Vol 9 (3) ◽

pp. e001877

Author(s):

Irfan N Bandey ◽

Jay R T Adolacion ◽

Gabrielle Romain ◽

Melisa Martinez Paniagua ◽

Xingyue An ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Single Cell ◽

Adoptive Cell Therapy ◽

Population Heterogeneity ◽

Serial Killer ◽

Car T Cells ◽

Car T ◽

Single Cell Profiling ◽

Killer T Cells

BackgroundAdoptive cell therapy based on the infusion of chimeric antigen receptor (CAR) T cells has shown remarkable efficacy for the treatment of hematologic malignancies. The primary mechanism of action of these infused T cells is the direct killing of tumor cells expressing the cognate antigen. However, understanding why only some T cells are capable of killing, and identifying mechanisms that can improve killing has remained elusive.MethodsTo identify molecular and cellular mechanisms that can improve T-cell killing, we utilized integrated high-throughput single-cell functional profiling by microscopy, followed by robotic retrieval and transcriptional profiling.ResultsWith the aid of mathematical modeling we demonstrate that non-killer CAR T cells comprise a heterogeneous population that arise from failure in each of the discrete steps leading to the killing. Differential transcriptional single-cell profiling of killers and non-killers identified CD137 as an inducible costimulatory molecule upregulated on killer T cells. Our single-cell profiling results directly demonstrate that inducible CD137 is feature of killer (and serial killer) T cells and this marks a different subset compared with the CD107apos (degranulating) subset of CAR T cells. Ligation of the induced CD137 with CD137 ligand (CD137L) leads to younger CD19 CAR T cells with sustained killing and lower exhaustion. We genetically modified CAR T cells to co-express CD137L, in trans, and this lead to a profound improvement in anti-tumor efficacy in leukemia and refractory ovarian cancer models in mice.ConclusionsBroadly, our results illustrate that while non-killer T cells are reflective of population heterogeneity, integrated single-cell profiling can enable identification of mechanisms that can enhance the function/proliferation of killer T cells leading to direct anti-tumor benefit.

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Single-cell profiling identifies pre-existing CD19-negative subclones in a B-ALL patient with CD19-negative relapse after CAR-T therapy

Nature Communications ◽

10.1038/s41467-021-21168-6 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Tracy Rabilloud ◽

Delphine Potier ◽

Saran Pankaew ◽

Mathis Nozais ◽

Marie Loosveld ◽

...

Keyword(s):

Targeted Therapy ◽

T Cell ◽

Single Cell ◽

Remission Rate ◽

Lymphoblastic Leukemia ◽

Leukemic Cells ◽

T Cell Therapy ◽

Car T Cell Therapy ◽

Car T ◽

Single Cell Profiling

AbstractChimeric antigen receptor T cell (CAR-T) targeting the CD19 antigen represents an innovative therapeutic approach to improve the outcome of relapsed or refractory B-cell acute lymphoblastic leukemia (B-ALL). Yet, despite a high initial remission rate, CAR-T therapy ultimately fails for some patients. Notably, around half of relapsing patients develop CD19 negative (CD19neg) B-ALL allowing leukemic cells to evade CD19-targeted therapy. Herein, we investigate leukemic cells of a relapsing B-ALL patient, at two-time points: before (T1) and after (T2) anti-CD19 CAR-T treatment. We show that at T2, the B-ALL relapse is CD19 negative due to the expression of a non-functional CD19 transcript retaining intron 2. Then, using single-cell RNA sequencing (scRNAseq) approach, we demonstrate that CD19neg leukemic cells were present before CAR-T cell therapy and thus that the relapse results from the selection of these rare CD19neg B-ALL clones. In conclusion, our study shows that scRNAseq profiling can reveal pre-existing CD19neg subclones, raising the possibility to assess the risk of targeted therapy failure.

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