Journal: bioRxiv

Article Title: Transcriptional Activation of Regenerative Hematopoiesis via Vascular Niche Sensing

doi: 10.1101/2023.03.27.534417

Figure Lengend Snippet: a, Schema for analysis of BM primitive SLAM LSK HSPCs. Murine femurs and tibias were harvested, flushed, and crushed, to collect maximal yield of bone and marrow cells. BM labeled cells were flow sorted for the SLAM LSK markers: Live\Ter-119 neg \Lineage neg \Sca-1 + \c-Kit + \CD150 + \CD48 neg . Next, combined multiome single-nuclei RNA/ATAC (snRNA/ATAC) sequencing analysis was performed. b, Weighted nearest neighbor (WNN) UMAP with hematopoietic stem and progenitor cell type annotation for hematopoietic stem cell (HSC), multi-potent progenitor (MPP), megakaryocyte progenitor (MkP), and erythrocyte progenitor (EryP) sub-cluster representation. c, Engrafting LTR-HSC transcriptional signature (from Rodriguez-Fraiticelli et al . 2020) assigned on a WNN UMAP space. d, Heatmap representation of differential transcriptional nuclei output from distinct HSPC sub-clusters by averaged Z-score, with selected genes presented. e, Heatmap representation of differential ChromVAR motif activity in distinct HSPC sub-clusters by averaged Z-score, with selected TF motifs presented.

Article Snippet: Human hematopoietic CD34 + HSPCs from either cord blood (CB) or mobilized peripheral blood (mPB) donors, were isolated using human CD34 microbead kit (Miltenyi Biotec) and by passing twice through LS columns (Miltenyi Biotec) attached to a magnetic stand, achieving a purity >95%.

Techniques: Labeling, Sequencing, Activity Assay

Journal: bioRxiv

Article Title: Transcriptional Activation of Regenerative Hematopoiesis via Vascular Niche Sensing

doi: 10.1101/2023.03.27.534417

Figure Lengend Snippet: WT, Fli-1 ROSAΔ , and Fli-1 ROSAΔ with conditionally inducible Notch1 internal component overexpression transgene (Fli-1 ROSAΔ N1-IC iOE ) BM LSK HSPCs were isolated and expanded as described in Fig. S5A. Harvested cells were analyzed by flow cytometry and/or flow sorted for LSK HSPCs which were competitively co-transplanted with congenic SJL BM cells into lethally irradiated congenic SJL recipient mice. a, Representative images of co-cultures at the end point before harvest. Yellow arrows indicate megakaryocytes. Note expansion of round hematopoietic cells in Fli-1 ROSAΔ N1-IC iOE co-cultures without any appearance of megakaryocytes. Bar = 100 µM. b, Frequency of LSK HSPCs was determined by flow cytometry and fold expansion was calculated. One-way ANOVA multiple comparisons was used; n=4 BM donor mice per genotype, with 2 technical replicates per donor. c, Frequency of chimerism indicating engraftment levels as determined by flow cytometry. One-way ANOVA multiple comparisons was used; n=8 recipient mice per genotype. d-g, WT, Fli-1 ROSAΔ , and Fli-1 ROSAΔ with conditionally inducible Notch1 internal component overexpression transgene (Fli-1 ROSAΔ N1-IC iOE ) BM LSK HSPCs were isolated and expanded. After 48 hours of expansion in co-culture, cells were harvested and hematopoietic LSK HSPCs were sorted and applied for single cell RNA-seq analysis; n=4 per genotype “pooled” together. d, Dimensionality reduction by UMAP of single cell transcriptomes from co-cultured and sorted LSK HSPCs. e. Dot plot for the E-SLAM HSC markers EPCR, CD34, and CD150 per cluster identity. Dot plot size indicates percent expression in cluster and color intensity indicates average expression score. f. Distribution of clusters 7 and 15 in UMAP for HSC identified populations among LSK HSPC clusters based on Garnette and the Dot plot from panel (e). g, Dot plot for cell cycle scores (S phase and G2/M phases) per cluster identity. Dot plot size indicates percent expression in cluster and color intensity indicates average expression score. h, Cell cycle status classification in UMAP for clusters 7 and 15: WT (left UMAP), Fli-1 ROSAΔ (middle UMAP) and Fli1 ROSAΔ N1-IC iOE (right UMAP). i-l, BM cells were harvested, 16 weeks post engraftment of WT and “rescued” Fli-1 ROSAΔ N1-IC iOE long term repopulating HSCs and pooled from n=4 recipient mice (from 4 different donors) per genotype. CD45.2 + /Lin − cells were flow sorted and applied for single cell RNA-seq analysis. (HSC: hematopoietic stem cells, MPP: multi-potent progenitors, LPP: lymphoid-primed progenitors, MEP: megakaryocyte/erythroid progenitors). i, Dimensionality reduction by UMAP of single cell transcriptomes from donor-derived lineage-negative BM samples (CD45.2 + /Lin − ) following transplantation of WT or Fli1 ROSAΔ N1-IC iOE LSK HSPC. j, Cell type classification of hematopoietic sub-populations using Garnette: WT (left panel) and Fli1 ROSAΔ N1-IC iOE (right panel) samples in UMAP, and distribution of cell types between samples (bar plot). k, Distribution of WT and Fli1 ROSAΔ N1-IC iOE sample cells in UMAP among populations classified as multi-lineage stem/progenitor cell types (HSC, MPP, or LPP) using Garnette, and distribution of cell types between samples (bar plot). See also Table S5. l , Heatmaps of gene-set scores for the subset of multi-lineage stem and progenitor cell types (HSC, MPP, LPP) in UMAP (left panels), and violin plots of gene-set scores between samples (WT vs Fli1 ROSAΔ N1-IC iOE ) (right panels), for HSC molecular overlap signature genes (from Wilson et al . 2015) and engrafting long term HSC signature genes (from Rodriguez-Fraiticelli et al . 2020). Gene-set scores in violin plots are shown only for the subset of cells classified as HSC cell type. Wilcoxon Rank Sum Test was used to determine p-Values.

Article Snippet: Human hematopoietic CD34 + HSPCs from either cord blood (CB) or mobilized peripheral blood (mPB) donors, were isolated using human CD34 microbead kit (Miltenyi Biotec) and by passing twice through LS columns (Miltenyi Biotec) attached to a magnetic stand, achieving a purity >95%.

Techniques: Over Expression, Isolation, Flow Cytometry, Irradiation, Co-Culture Assay, RNA Sequencing, Cell Culture, Expressing, Derivative Assay, Transplantation Assay

Journal: bioRxiv

Article Title: Transcriptional Activation of Regenerative Hematopoiesis via Vascular Niche Sensing

doi: 10.1101/2023.03.27.534417

Figure Lengend Snippet: a-d, Sorted CD45 + /CD34 + HSPC from cord blood (CB) and adult mobilized peripheral blood (mPB) sources, were co-cultured on top of a vascular niche for 48h to encourage HSPC activation. Next, cells were harvested and CD45 + /CD34 + HSPC were sorted and applied for single cell RNA-seq analysis. a-c, Dimensionality reduction by UMAP of single cell transcriptomes from co-cultured and sorted CD45 + /CD34 + HSPC, displaying cell distribution by CB and mPB source (a), cluster identity (b), and single cell cycle phase (c). d, Dot plot for cell cycle scores (S phase and G2/M phases) per cellular source (CB or mPB), for total cells in analysis (upper panel) and for HSC\MPP cluster (lower panel). Dot plot size indicates percent expression in cluster and color intensity indicates average expression score. e, To highly enrich for HSCs, the mononuclear fraction from CB (n=3) and mPB (n=4) sources was isolated, labeled, and sorted for CD45 + /CD34 + /CD38 − /CD45RA − /CD90 + /CD49f + . Sorted cells were lysed and processed for RNAseq analysis. GSEA analysis plots for HSC activation signatures (left panels) and for HSC quiescence signatures (right panels) acquired from Venezia et al., 2004 (upper panels) and from Roy et al., 2021 (lower panels), showing a positive enrichment for the activation signatures in CB HSCs and positive enrichment for quiescence signatures in mPB HSCs. f, g, UMAP projections of the single cell ATAC-seq enrichment analysis for sorted HSPCs of all 10 signatures identified in Takayama et al., 2021. Colors indicate the degree of enrichment in each cell (blue, depleted; red, enriched). Scale bar indicates enrichment Z-score. f, Enrichment for activated HSPCs (upper panel) and quiescent HSPCs (lower panel) signatures defined in in Takayama et al., 2021., as calculated by chromVAR. g, Enrichment for called peaks from the FLI-1 HSPC ChIP-seq (Fli-1 signature) data set (Beck et al., 2013) overlayed on single cell ATAC-seq HSPC data set (Takayama et al., 2021), as analyzed by chromVAR.

Article Snippet: Human hematopoietic CD34 + HSPCs from either cord blood (CB) or mobilized peripheral blood (mPB) donors, were isolated using human CD34 microbead kit (Miltenyi Biotec) and by passing twice through LS columns (Miltenyi Biotec) attached to a magnetic stand, achieving a purity >95%.

Techniques: Cell Culture, Activation Assay, RNA Sequencing, Expressing, Isolation, Labeling, ChIP-sequencing

Journal: bioRxiv

Article Title: Transcriptional Activation of Regenerative Hematopoiesis via Vascular Niche Sensing

doi: 10.1101/2023.03.27.534417

Figure Lengend Snippet: a, Schema of experimental design. Sorted human CD34 + HSPCs from cord blood (CB) or mobilized peripheral blood (mPB) sources were transduced by electroporation with FLI-1 modified-RNA molecules (2 µg FLI-1 modRNA per 10 5 cells) and expanded for 1 week on top of E4orf1 vascular niche cells in a sub-optimal ratio of 1:3 (HSPCs:ECs). Next, expansion co-cultures were analyzed and transplanted into immunodeficient NSG KitW41 mice without myeloablative preconditioning. b , Fold expansion of CB derived hematopoietic subtypes after 1 week in co-culture following transduction with FLI-1 (red) or control (blue) modified-RNA. Unpaired two tailed t-test was used; n = 4 CB donors. Each mark represents the averaged triplicate (n = 3 technical repeats) per donor. c , Fold expansion of mPB derived hematopoietic subtypes after 1 week in co-culture following transduction with FLI-1 (red) or control (blue) modified-RNA. Unpaired two tailed t-test was used; n = 4 mPB donors. Each mark represents the averaged triplicate (n = 3 technical repeats) per donor. d, Representative flow dot plots of mPB HSPC analysis post 1 week of expansion in co-culture following transduction with FLI-1 or control modified-RNA. The population of CD34 + \CD38 neg HSPCs is colored in red. e, Human CD45 chimerism analysis in peripheral blood (PB), spleen, and bone marrow (BM) of NSG KitW41 mice. Tissues were harvested and analyzed by flow cytometry 16 weeks post transplantation. Unpaired two tailed t-test was used; n = 6 mPB donors. f, Frequency of BM engrafted human HSPCs as determined by flow cytometry 16 weeks post transplantation. Unpaired two tailed t-test was used; n = 6 mPB donors. g, Representative flow dot plots for BM engrafted human CD34 + and CD34 + \CD38 neg HSPCs acquired 16 weeks post transplantation. Unpaired two tailed t-test was used; n = 6 mPB donors. h, Total BM from primary NSG KitW41 recipient mice was transplanted into secondary NSG KitW41 recipient mice without myeloablative preconditioning. Human CD45 chimerism in the BM of recipient mice was determined by flow cytometry 16 weeks post secondary transplantation. Unpaired two tailed t-test was used; n = 6 mPB donors.

Article Snippet: Human hematopoietic CD34 + HSPCs from either cord blood (CB) or mobilized peripheral blood (mPB) donors, were isolated using human CD34 microbead kit (Miltenyi Biotec) and by passing twice through LS columns (Miltenyi Biotec) attached to a magnetic stand, achieving a purity >95%.

Techniques: Electroporation, Modification, Derivative Assay, Co-Culture Assay, Transduction, Control, Two Tailed Test, Flow Cytometry, Transplantation Assay

Journal: bioRxiv

Article Title: Tim-3 co-stimulation promotes short-term effector T cells, restricts memory precursors and is dispensable for T cell exhaustion

doi: 10.1101/179002

Figure Lengend Snippet: A , CD25-depleted T cells from Nur77 GFP mice were stimulated for three days with anti-CD3/CD28 mAbs, followed by a seven day rest with IL-2. After three rounds of stimulation, cells were stained for PD-1 and Tim-3 and analyzed by flow cytometry. Representative of 5 technical replicates per experiment, repeated with n=5 mice. B , WT C57BL/6 mice were infected with LCMV Arm. After 30d, spleens were harvested for flow cytometry (n=5 mice, mean ± SD). representative of three independent experiments C , Tim-3 + vs. Tim-3 − CD8 + cells were further analyzed for expression of activation and differentiation markers shown in the histograms. D , splenocytes from the same experiments as panels b-c were stained with LCMV tetramers plus α Tim-3. (n=5 mice, mean ± SD) representative of three independent experiments. **p<0.01 by two-tailed paired Student’s t test. E , C57Bl/6 mice previously infected with LCMV-Arm (>30d.p.i.) were challenged with LM-GP33 and Tet − vs. Tet + CD8 + splenocytes were analyzed for KLRG1 and Tim-3 four days post-challenge. Representative of three independent experiments (n=5).

Article Snippet: CD25 depletion was done using a CD25 microbead kit (Miltenyi).

Techniques: Staining, Flow Cytometry, Infection, Expressing, Activation Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Effects of Post-Myocardial Infarction Heart Failure on the Bone Vascular Niche

doi: 10.1101/2020.05.29.123711

Figure Lengend Snippet: ( a, b ) Flow cytometry analysis of healthy and HF patient bone marrow. ( a ) Gating strategy for endothelial cells. Representative flow cytometry dot plots showing EC subsets with distinct expression of CD31 and Endomucin (EMCN) in healthy and HF bone marrow aspirates (gated on CD45 neg Lin neg viable single cells). ( b ) Type H endothelial cells are reduced in the HF patients compared to healthy controls (left panel), while the total number of endothelial cells remains unchanged (right panel) (N=8 for healthy, N=18 for HF patients). Data are shown as mean ± SEM. P-value was calculated by unpaired, Mann Whitney test. ( c - i ) scRNA-seq of a post-MI heart failure and an aged-matched healthy control. ( c , d ) Clustered cells from both subjects are displayed in t-SNE plots, colored by cluster (left), cell annotation (middle) and health status (right). ( d ) Expression of EMCN and PECAM1. EMCN is enriched in the cells corresponding to cluster 0. ( e - f ) Analysis of EMCN enriched cell cluster 0 population. ( e ) Dichotomization in EMCN enriched population shown in t-SNE plot (Left). Relative expression of key genes in the EMCN enriched population represented by features plots as indicated. ( f ) Violin plots showing the relative expression of key genes in the EMCN enriched population, confirming the significantly increased expression of IL1B and MYC in the HF patient. ( g ) Distribution of cells along pseudotime trajectory branchpoints. Pseudotime analysis revealed 13 states. ( h ) Distribution of cells among pseudotime states and relative IL1B expression. Distribution analysis revealed that states 10, 11, 12, and 13 are mainly populated by HF patient cells. IL1B expression is higher in states 12 and 13. Dashed line indicates normalized Unique Molecular Identifier (nUMI) counts of 2.5. ( i ). Gene Ontology term ranking of upregulated genes in pseudotime state 13.

Article Snippet: Lineage negative and CD31 positive BMCs were isolated using first immunomagnetic Lineage Cell Depletion Kit (130-092-211, Miltenyi Biotec) followed by positive selection with CD31 MicroBead Kit (130-091-935, Miltenyi Biotec) using a magnetic cell separation device (QuadroMACS Separator;130-090-976, Miltenyi Biotec).

Techniques: Flow Cytometry, Expressing, MANN-WHITNEY, Control

Journal: The Journal of Experimental Medicine

Article Title: High-efficiency nonviral CRISPR/Cas9-mediated gene editing of human T cells using plasmid donor DNA

doi: 10.1084/jem.20211530

Figure Lengend Snippet: Plasmid-based donor templates enable efficient nonviral gene editing of TRAC locus in primary T cells. (A–C) Titration of linear dsDNA donor template. (A) Diagram of linear dsDNA knock-in construct TRAC -mNG. (B) Bar graphs depicting knock-in efficiency, cell viability, total cell recovery, and edited cell recovery (mNG-positive cells) 3 d after electroporation with 1, 2, 4, 6, or 8 µg of linear dsDNA donor template together with Cas9-RNP targeting the TRAC locus. Circles represent individual donors; bars represent median values with range ( n = 4). (C) Representative contour plots showing the frequency of CD8 + T cells expressing mNG. (D–F) Titration of pUC57 plasmid donor template. (D) Diagram of pUC57 knock-in construct TRAC -mNG. (E) Bar graphs showing the frequency of CD8 + T cells expressing mNG, cell viability, total cell recovery, and edited cell recovery (mNG-positive cells) 3 d after electroporation with 1, 2, 4, 6, or 8 µg of pUC57 plasmid donor template together with Cas9-RNP targeting the TRAC locus. Circles represent individual donors; bars represent median values with range ( n = 4). (F) Representative contour plots showing the frequency of CD8 + T cells expressing mNG. (G–I) Titration of nanoplasmid donor template. (G) Diagram of nanoplasmid knock-in construct TRAC -mNG. (H) Bar graphs showing the frequency of CD8 + T cells expressing mNG, total cell recovery, and edited cell recovery (mNG-positive cells) 3 d after electroporation with 1, 2, 4, 6, or 8 µg of nanoplasmid donor template together with Cas9-RNP targeting the TRAC locus. Circles represent individual donors; bars represent median values with range ( n = 4). (I) Representative contour plots showing the frequency of CD8 + T cells expressing mNG. This experiment was performed twice. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 in RM one-way ANOVA with Geisser–Greenhouse correction.

Article Snippet: Primary human CD8 + and CD4 + T cells were isolated by positive selection from buffy coats using the StraightFrom Buffy Coat CD8 MicroBead Kit or CD4 MicroBead Kit, respectively, according to the manufacturer’s instructions (Miltenyi Biotec).

Techniques: Plasmid Preparation, Titration, Knock-In, Construct, Cell Recovery, Electroporation, Expressing

Journal: The Journal of Experimental Medicine

Article Title: High-efficiency nonviral CRISPR/Cas9-mediated gene editing of human T cells using plasmid donor DNA

doi: 10.1084/jem.20211530

Figure Lengend Snippet: Optimization of nonviral gene editing in primary T cells using plasmid-based donor templates. (A–F) Titration of linear dsDNA and nanoplasmid donor templates in CD8 + T cell cultures in RPMI/10% FBS medium. (A) Diagram of linear dsDNA knock-in construct TRAC -mNG. (B) Representative contour plots showing the frequency of CD8 + T cells expressing mNG. (C) Bar graphs depicting knock-in efficiency, cell viability, total cell recovery, and edited cell recovery (mNG-positive cells) of CD8 + T cells cultured in RPMI/10% FBS 3 d after electroporation with 1, 2, or 4 µg of linear dsDNA donor template together with Cas9-RNP targeting the TRAC locus. Circles represent individual donors; bars represent median values with range ( n = 4). This experiment was performed twice. (D) Diagram of nanoplasmid knock-in construct TRAC -mNG. (E) Representative contour plots showing the frequency of CD8 + T cells expressing mNG. (F) Bar graphs depicting knock-in efficiency, cell viability, total cell recovery, and edited cell recovery (mNG-positive cells) of CD8 + T cells cultured in RPMI/10% FBS 3 d after electroporation with 1, 2, or 4 µg of nanoplasmid donor template together with Cas9-RNP targeting the TRAC locus. Circles represent individual donors; bars represent median values with range ( n = 4). This experiment was performed twice. (G) Bar graphs depicting knock-in efficiency, cell viability, total cell recovery, and edited cell recovery 3 d after electroporation with 2 µg of either linear dsDNA or nanoplasmid donor template together with Cas9-RNP targeting the TRAC locus in the presence of absence of PGA. Circles represent individual donors; bars represent median values with range ( n = 3). This experiment was performed twice. (H) Bar graphs depicting knock-in efficiency, cell viability, total cell recovery, and edited cell recovery 3 d after electroporation with 2 µg of either linear dsDNA or nanoplasmid donor template that either did or did not contain truncated Cas9 target sequences (tCTS) together with Cas9-RNP targeting the TRAC locus. Circles represent individual donors; bars represent median values with range ( n = 3). This experiment has been performed twice. *, P < 0.05; **, P < 0.01; ***, P < 0.001 in RM one-way ANOVA with Geisser–Greenhouse correction (C and F) or paired t test (G and H).

Article Snippet: Primary human CD8 + and CD4 + T cells were isolated by positive selection from buffy coats using the StraightFrom Buffy Coat CD8 MicroBead Kit or CD4 MicroBead Kit, respectively, according to the manufacturer’s instructions (Miltenyi Biotec).

Techniques: Plasmid Preparation, Titration, Knock-In, Construct, Expressing, Cell Recovery, Cell Culture, Electroporation

Journal: The Journal of Experimental Medicine

Article Title: High-efficiency nonviral CRISPR/Cas9-mediated gene editing of human T cells using plasmid donor DNA

doi: 10.1084/jem.20211530

Figure Lengend Snippet: Cytokine production and stress response induced in T cells following exposure to dsDNA donor templates. (A) IFN-α measured by Simoa and IFN-γ, TNF-α, and IL-2 measured by Luminex from CD8 + T cells 18 h after transfection with Cas9-RNP targeting the TRAC locus alone or together with nanoplasmid donor template compared with non-transfected control T cells (No RNP). Circles represent individual donors; bars represent median values with range ( n = 4). This experiment was performed once for Simoa and twice for Luminex. (B) GSEA from RNA-sequencing of CD8 + T cells after transfection with Cas9-RNP targeting the TRAC with nanoplasmid donor template compared with Cas9-RNP alone. Gene sets for IFN-γ response, IFN-α response, TNF-α signaling, and inflammatory response were significantly enriched. (C) GSEA from RNA-seq of CD8 + T cells after transfection with Cas9-RNP targeting the TRAC with linear dsDNA donor template compared to Cas9-RNP alone. Gene sets for IFN-γ response, IFN-α response, TNF-α signaling, and inflammatory response were significantly enriched. (B and C) The y axis represents enrichment score, and on the x axis are genes (vertical black lines) represented in gene sets. The colored band at the bottom represents the degree of differentially expressed genes (red for upregulation and blue for downregulation). (D) Gene set enrichment analysis of all 375 upregulated genes in both Nanoplasmid/Cas9-RNP and linear dsDNA/Cas9-RNP over Cas9-RNP-only using the GSEA MSigDB Hallmark 2020. (E–H) Heatmaps showing upregulated genes in Nanoplasmid/Cas9-RNP and linear dsDNA/Cas9-RNP over Cas9-RNP-only that mostly contributed to IFN-α response (E), TNF-α response (F), apoptosis (G), or inflammatory response (H; all MSigDB Hallmark). Color-coded by the normalized RNA-seq count data with variance stabilizing transformation (VST). This experiment was performed once. *, P < 0.05; **, P < 0.01; ****, P < 0.0001 in one-way ANOVA.

Article Snippet: Primary human CD8 + and CD4 + T cells were isolated by positive selection from buffy coats using the StraightFrom Buffy Coat CD8 MicroBead Kit or CD4 MicroBead Kit, respectively, according to the manufacturer’s instructions (Miltenyi Biotec).

Techniques: Luminex, Transfection, Control, RNA Sequencing, Transformation Assay

Journal: The Journal of Experimental Medicine

Article Title: High-efficiency nonviral CRISPR/Cas9-mediated gene editing of human T cells using plasmid donor DNA

doi: 10.1084/jem.20211530

Figure Lengend Snippet: Optimization of CRISPR/Cas9-mediated gene knock-in with plasmid-based donor DNA in CD4 + and CD8 + T cells. (A and B) Homology arm optimization for plasmid-based donor templates. (A) Representative contour plots showing the frequency of CD8 + T cells expressing mNG. (B) Bar graphs depicting knock-in efficiency, cell viability, total cell recovery, and edited cell recovery (mNG-positive cells) 3 d after electroporation with pUC57 plasmid or nanoplasmid donor templates with homology arm lengths between 100 bp and 2,000 bp (amounts equimolar to 4 µg of the pUC57 2,000 bp construct) together with Cas9-RNP targeting the TRAC locus ( n = 2). Circles represent individual donors; bars represent median values with range. This experiment was performed three times. (C) Frequency of CD8 + T cells expressing mNG, cell viability, total cell recovery, and edited cell recovery (mNG-positive cells) 3 d after electroporation after stimulating cells for 24, 36, 48, or 72 h prior to electroporation with nanoplasmid donor template together with Cas9-RNP targeting the TRAC locus ( n = 4). Circles represent individual donors; bars represent median values with range. This experiment was performed twice. (D) Nucleofection pulse code optimization in CD8 + T cells electroporated with nanoplasmid donor template and Cas9-RNP targeting the TRAC locus. Graph shows frequency of cells expressing mNG and edited cell recovery (mNG-positive cells) 3 d after electroporation. Each circle represents a distinct pulse code. Data are representative of three independent CD8 + T cell donors. This experiment was performed twice. (E and F) Gene editing targeting the TRAC locus in CD4 + T cells. Representative contour plot showing the frequency of CD4 + T cells expressing mNG (E) and bar graphs (F) depicting knock-in efficiency, cell viability, total cell recovery, and edited cell recovery (mNG-positive cells) 5 d after electroporation of CD4 + T cells with TRAC -mNG nanoplasmid donor template together with Cas9-RNP targeting the TRAC locus ( n = 3). Circles represent individual donors; bars represent median values with range. This experiment was performed twice. *, P < 0.05; **, P < 0.01 in RM one-way ANOVA with Geisser–Greenhouse correction.

Article Snippet: Primary human CD8 + and CD4 + T cells were isolated by positive selection from buffy coats using the StraightFrom Buffy Coat CD8 MicroBead Kit or CD4 MicroBead Kit, respectively, according to the manufacturer’s instructions (Miltenyi Biotec).

Techniques: CRISPR, Gene Knock-In, Plasmid Preparation, Expressing, Knock-In, Cell Recovery, Electroporation, Construct

Journal: The Journal of Experimental Medicine

Article Title: High-efficiency nonviral CRISPR/Cas9-mediated gene editing of human T cells using plasmid donor DNA

doi: 10.1084/jem.20211530

Figure Lengend Snippet: Nonviral TCR editing using plasmid DNA donors. (A) Diagram of TCR α and β genomic loci. V gene (purple), D gene (red), J gene (blue), and constant region (green) segments. sg TRAC and sg TRBC targeting sites are indicated. (B) Diagrams of nanoplasmid knock-in constructs TRAC -1G4TCR, TRAC -TCR6-2, and TRAC -CD19CAR. (C, E, and G) Representative contour plots (left) and bar graphs (right) showing the frequencies of CD8 + T cells expressing (C) a NY-ESO-1-specific 1G4 TCR, (E) a CMV-specific pp65 6-2 TCR, and (G) a CD19-CAR 5 d after electroporation using nanoplasmid donor templates together with Cas9-RNPs targeting the TRAC locus. (D, F, and H) Bar graphs showing the cell viability, total cell recovery, and edited cell recovery 5 d after electroporation using nanoplasmid donor templates encoding (D) a NY-ESO-1–specific 1G4 TCR, (F) a CMV-specific pp65 6-2 TCR, and (H) a CD19-CAR together with Cas9-RNPs targeting the TRAC locus. Circles represent individual donors; bars represent median values with range ( n = 3). This experiment was performed three times. (I) Lactate levels in culture supernatant analyzed by luminescence using the Lactate-Glo Assay were measured 1, 3, 5, and 7 d after transfection of CD8 + T cells with sg TRAC /sg TRBC Cas9-RNP (RNP only) or sg TRAC /sg TRBC Cas9-RNP and nanoplasmid donor template targeting the TRAC locus (RNP + nanoplasmid) compared with non-transfected control T cells (No RNP); RLU, relative light units. (J) Number of cells recovered from cultures 7 d after transfection of CD8 + T cells with sg TRAC /sg TRBC Cas9-RNP (RNP only) or sg TRAC /sg TRBC Cas9-RNP and nanoplasmid donor template targeting the TRAC locus (RNP + nanoplasmid) compared with non-transfected control T cells (No RNP). This experiment was performed three times. *, P < 0.05 in RM one-way ANOVA with Geisser–Greenhouse correction.

Article Snippet: Primary human CD8 + and CD4 + T cells were isolated by positive selection from buffy coats using the StraightFrom Buffy Coat CD8 MicroBead Kit or CD4 MicroBead Kit, respectively, according to the manufacturer’s instructions (Miltenyi Biotec).

Techniques: Plasmid Preparation, Knock-In, Construct, Expressing, Electroporation, Cell Recovery, Glo Assay, Transfection, Control

Journal: The Journal of Experimental Medicine

Article Title: High-efficiency nonviral CRISPR/Cas9-mediated gene editing of human T cells using plasmid donor DNA

doi: 10.1084/jem.20211530

Figure Lengend Snippet: Nonviral TCR editing in CD4 + and CD8 + T cells using plasmid DNA donors. (A) TCR expression on the cell surface by flow cytometry of CD8 + T cells 48 h after transfection with Cas9-RNP targeting the TRAC (sg TRAC ) or TRBC (sg TRBC ) loci. Circles represent individual donors; bars represent median values with range ( n = 3). This experiment was performed three times. (B–G) TCR editing in CD4 + T cells. Representative contour plots showing the frequencies of CD4 + T cells expressing a NY-ESO-1-specific 1G4 TCR (B), a CMV-specific pp65 6-2 TCR (D), and a CD19-CAR (F) and bar graphs showing the knock-in efficiency and cell viability 5 d after electroporation using nanoplasmid donor templates encoding a NY-ESO-1-specific 1G4 TCR (C), a CMV-specific pp65 6-2 TCR (E), and a CD19-CAR (G) together with Cas9-RNPs targeting the TRAC locus. Circles represent individual donors; bars represent median values with range ( n = 4). This experiment was performed twice. (H and I) Diagram depicting all possible translocation events between the TRAC , TRBC1 , and TRBC2 genomic loci (H). Bar graph (I) showing the frequencies of individual translocation events between the TRAC , TRBC1 , and TRBC2 genomic loci quantified by ddPCR in CD8 + T cells co-transfected with Cas9-RNPs targeting the TRAC and TRBC loci or in non-transfected control T cells. Circles represent individual donors; bars represent median values with range ( n = 4). This experiment was performed twice. (J and K) Representative histograms (J) and bar graphs (K) showing proportions of CD137-expressing pp65 TCR knock-in CD8 + T cells stimulated with indicated concentrations of pp65 495–503 peptide. Circles represent individual donors; bars represent median values with range ( n = 3). This experiment was performed twice. (L) Bar graphs showing IFN-γ and TNF-α production by pp65 TCR knock-in CD8 + T cells stimulated with indicated concentrations of pp65 495–503 peptide. Circles represent individual donors; bars represent median values with range ( n = 3). This experiment was performed twice. (M) Representative histograms showing the frequencies of CFSE-positive target cells and CFSE-negative reference cells in co-cultures with pp65 TCR knock-in CD8 + T cells in the absence or presence of the cognate peptide. (N) Graphs showing specific lysis calculated in the absence of peptide or with 0.1 µM of pp65 495–503 peptide. Circles represent individual donors; bars represent median values with range ( n = 3). This experiment was performed twice. (O) Bar graphs showing IFN-γ and TNF-α production by TCR6-2 (irrelevant TCR) or CD19-CAR knock-in CD4 + T cells from two donors (D1 and D2) in co-cultures with CD19-expressing B cells. Circles represent technical replicates; bars represent median values with range ( n = 9). This experiment was performed twice. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 in RM one-way ANOVA with Geisser–Greenhouse correction (A and K), paired t test (N), and one-way ANOVA (O).

Article Snippet: Primary human CD8 + and CD4 + T cells were isolated by positive selection from buffy coats using the StraightFrom Buffy Coat CD8 MicroBead Kit or CD4 MicroBead Kit, respectively, according to the manufacturer’s instructions (Miltenyi Biotec).

Techniques: Plasmid Preparation, Expressing, Flow Cytometry, Transfection, Knock-In, Electroporation, Translocation Assay, Control, Lysis

Journal: The Journal of Experimental Medicine

Article Title: High-efficiency nonviral CRISPR/Cas9-mediated gene editing of human T cells using plasmid donor DNA

doi: 10.1084/jem.20211530

Figure Lengend Snippet: TCR-engineered T cells recognize and kill antigen-expressing target cells. (A and B) Representative histograms (A) and bar graphs (B) showing proportion of CD137 expression of 1G4 TCR knock-in CD8 + T cells stimulated with indicated concentrations of NY-ESO-1 157–165 peptide. Circles represent individual donors; bars represent median values with range ( n = 4). This experiment was performed twice. (C and D) Bar graphs showing IFN-γ (C) or TNF-α (D) production by 1G4 TCR knock-in CD8 + T cells stimulated with indicated concentrations of NY-ESO-1 157–165 peptide. Circles represent individual donors; bars represent median values with range ( n = 4). This experiment was performed twice. (E) Representative histograms showing the frequencies of CFSE-positive target cells and CFSE-negative reference cells in co-cultures with 1G4 TCR knock-in CD8 + T cells in the absence or presence of the cognate peptide. (F) Graphs showing specific lysis calculated in the absence of peptide or with 0.1 µM of NY-ESO-1 157–165 peptide. Circles represent individual donors; bars represent median values with range ( n = 4). This experiment was performed twice. (G) Bar graphs showing IFN-γ, TNF-α, and granzyme B (GzmB) production by TCR knock-out or 1G4 TCR knock-in CD8 + T cells from three donors co-cultured with A-375 cells that express the NY-ESO-1 antigen. Circles represent technical replicates; bars represent median values with range ( n = 3). This experiment was performed twice. (H) Representative images for A-375 cells that express the NY-ESO-1 antigen and were labeled with a cytoplasmic dye and co-cultured with TCR knock-out CD8 + T cells (left) or 1G4 TCR knock-in CD8 + T cells (right) 2 and 18 h after culture seeding in the presence of caspase 3/7-green apoptosis reagent. Scale bars indicate 300 µm distance. (I) Representative target cell killing over time as measured by the Cas3/7-positive object count in co-cultures of A-375 cells expressing the NY-ESO-1 antigen and labeled with a cytoplasmic dye and co-cultured with TCR knock-out CD8 + T cells (open circles) or 1G4 TCR knock-in CD8 + T cells (filled circles). Mean values ± SD of six technical replicates. This experiment was performed twice with three independent donors per experiment. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 in RM one-way ANOVA with Geisser–Greenhouse correction (B–D); paired t test (F); one-way ANOVA (G); or Tukey’s multiple comparisons test, two-way ANOVA (I).

Article Snippet: Primary human CD8 + and CD4 + T cells were isolated by positive selection from buffy coats using the StraightFrom Buffy Coat CD8 MicroBead Kit or CD4 MicroBead Kit, respectively, according to the manufacturer’s instructions (Miltenyi Biotec).

Techniques: Expressing, Knock-In, Lysis, Knock-Out, Cell Culture, Labeling

Journal: The Journal of Experimental Medicine

Article Title: High-efficiency nonviral CRISPR/Cas9-mediated gene editing of human T cells using plasmid donor DNA

doi: 10.1084/jem.20211530

Figure Lengend Snippet: Kinetics of gene expression following transient transfection of linear dsDNA, plasmid, and nanoplasmid. (A) Diagram of nanoplasmid knock-in construct RAB11A -YFP. (B and C) Representative histograms showing the frequencies of CD8 + T cells expressing YFP (B) and bar graphs (C) depicting frequency of YFP expression, cell viability, total cell recovery, and edited cell recovery 3, 5, or 7 d after electroporation with promoter-containing nanoplasmid donor template together with (red) or without (blue) Cas9-RNPs targeting the RAB11A locus. Circles represent technical replicates; bars represent median values with range ( n = 3). This experiment was performed twice. (D) Diagram of linear dsDNA knock-in construct RAB11A -YFP. (E and F) Representative histograms showing the frequencies of CD8 + T cells expressing YFP (E) and bar graph (F) depicting frequency of YFP expression 3, 5, or 7 d after electroporation with promoter-containing linear dsDNA donor templates together with (red) or without (blue) Cas9-RNPs targeting the RAB11A locus. Circles represent technical replicates; bars represent median values with range ( n = 3). This experiment was performed once. (G) Diagram of pUC57 plasmid knock-in construct RAB11A -YFP. (H and I) Representative histograms showing the frequencies of CD8 + T cells expressing YFP (H) and bar graph (I) depicting frequency of YFP expression 3, 5, or 7 d after electroporation with promoter-containing pUC57 plasmid donor templates together with (red) or without (blue) Cas9-RNPs targeting the RAB11A locus. Circles represent technical replicates; bars represent median values with range ( n = 3). This experiment was performed twice. *, P < 0.05; **, P < 0.05; ***, P < 0.001 in Sidak’s multiple comparisons test with RM one-way ANOVA with Geisser–Greenhouse correction.

Article Snippet: Primary human CD8 + and CD4 + T cells were isolated by positive selection from buffy coats using the StraightFrom Buffy Coat CD8 MicroBead Kit or CD4 MicroBead Kit, respectively, according to the manufacturer’s instructions (Miltenyi Biotec).

Techniques: Gene Expression, Transfection, Plasmid Preparation, Knock-In, Construct, Expressing, Cell Recovery, Electroporation

Journal: The Journal of Experimental Medicine

Article Title: High-efficiency nonviral CRISPR/Cas9-mediated gene editing of human T cells using plasmid donor DNA

doi: 10.1084/jem.20211530

Figure Lengend Snippet: Generation of reporters of gene expression. (A) Diagram of nanoplasmid knock-in construct RAB11A -YFP. (B and C) Histogram overlay for YFP expression (B) and bar graphs (C) showing the frequency of YFP expression and cell viability of CD8 + T cells transfected with RAB11A -YFP nanoplasmid with or without RAB11A targeting Cas9-RNP 10 d after electroporation. Circles represent individual donors; bars represent median values with range ( n = 3). This experiment was performed three times. (D) Diagram of nanoplasmid knock-in construct AAVS1- mNG. (E and F) Histogram overlay for mNG expression (E) and bar graphs (F) showing the frequency of mNG expression and cell viability of CD8 + T cells transfected with AAVS1- mNG nanoplasmid with or without AAVS1 targeting Cas9-RNP 10 d after electroporation. Circles represent individual donors, and bars represent median values with range ( n = 4). This experiment was performed three times. (G) Diagram of nanoplasmid knock-in construct CD4- mNG. (H and I) Representative contour plots (H) and bar graphs (I) showing the frequency of CD4 + and CD8 + T cells expressing mNG and cell viability 10 d after electroporation of a nanoplasmid donor template and Cas9-RNP targeting the CD4 locus. Circles represent individual donors, and bars represent median values with range ( n = 4 for CD4 + T cells, n = 3 for CD8 + T cells). This experiment was performed twice. (J) Histogram overlay for CD4 expression in CD4 + T cells transfected with CD4- mNG nanoplasmid together with a non-targeting control Cas9-RNP (sgNTC) or a Cas9-RNP targeting the CD4 locus (sg CD4 ) 10 d after electroporation. (K) Diagrams of nanoplasmid knock-in constructs TNFRSF9 -mNG and RAB11A -YFP (left) and representative contour plots (right) showing the frequency of CD8 + T cells expressing CD137 and mNG after electroporation with a nanoplasmid mNG reporter construct targeting the TNFRSF9 locus or a constitutive YFP expressing construct targeting the RAB11A locus together with the respective Cas9-RNP either without restimulation or 6 h after restimulation with Transact. (L) Bar graphs showing the frequency of YFP (blue) and mNG (red) expressing CD8 + T cells over time after electroporation with a nanoplasmid mNG reporter construct targeting the TNFRSF9 locus or a constitutive YFP expressing construct targeting the RAB11A locus together with the respective Cas9-RNP and restimulation with Transact at time 0 h. Circles represent individual donors; bars represent median values with range ( n = 4). This experiment was performed twice. (M) Bar graphs showing the geometric mean fluorescent intensity (gMFI) of CD137 expression in CD8 + T cells over time after electroporation with a nanoplasmid mNG reporter construct targeting the TNFRSF9 locus or a constitutive YFP expressing construct targeting the RAB11A locus together with the respective Cas9-RNP and restimulation with Transact at time 0 h ( n = 4). Circles represent individual donors; bars represent median values with range. *, P < 0.05; **, P < 0.01 in paired t test (C, F, I, and J) or in RM one-way ANOVA with Geisser–Greenhouse correction (L).

Article Snippet: Primary human CD8 + and CD4 + T cells were isolated by positive selection from buffy coats using the StraightFrom Buffy Coat CD8 MicroBead Kit or CD4 MicroBead Kit, respectively, according to the manufacturer’s instructions (Miltenyi Biotec).

Techniques: Gene Expression, Knock-In, Construct, Expressing, Transfection, Electroporation, Control

Journal: The Journal of Experimental Medicine

Article Title: High-efficiency nonviral CRISPR/Cas9-mediated gene editing of human T cells using plasmid donor DNA

doi: 10.1084/jem.20211530

Figure Lengend Snippet: Multiplexed gene knock-in in human T cells. (A–C) Diagrams of nanoplasmid knock-in constructs are provided on the top. Representative contour plots (left) and bar graphs (right) showing the frequency of CD8 + T cells expressing mNG (A) 10 d after electroporation with a nanoplasmid TRAC -mNG donor template and Cas9-RNPs targeting the TRAC locus, mCherry (B) 10 d after electroporation with a nanoplasmid TRAC -mCherry donor template and Cas9-RNPs targeting the TRAC locus, or either mNG or mCherry (C) 10 d after electroporation with two nanoplasmid donor templates ( TRAC -mNG and TRAC -mCherry) and Cas9-RNPs targeting the TRAC locus. Graph on the right for C indicates proportion of transgene expressing cells that express mNG (green), mCherry (red), or both (blue). Circles represent individual donors; bars represent median values with range ( n = 3). This experiment was performed three times. (D–F) Diagrams of nanoplasmids used in dual targeting study, RAB11A -YFP and TRAC -mCherry (D); representative contour plot (E) showing the frequency of CD8 + T cells expressing YFP, mCherry, or both; and bar graphs (F) showing knock-in efficiency, cell viability, and total cell recovery of CD8 + T cells 10 d after electroporation with nanoplasmid donors RAB11A -YFP and TRAC -mCherry and Cas9-RNPs targeting the RAB11A and TRAC loci. (G) Proportion of transgene expressing T cells co-transfected with nanoplasmid donors RAB11A -YFP and TRAC -mCherry and Cas9-RNPs targeting the RAB11A and TRAC loci that express YFP (green), mCherry (red), or both (blue). Circles represent individual donors, and bars represent median values with range ( n = 4). This experiment was performed three times. (H) Diagrams of nanoplasmids used in dual targeting study, AAVS1 -mNG and TRAC -mCherry. (I and J) Representative contour plot showing the frequency of CD8 + T cells expressing mNG, mCherry or both (I) and bar graphs (J) showing knock-in efficiency, cell viability, and total cell recovery of CD8 + T cells 10 d after electroporation with nanoplasmid donors AAVS1 -mNG and TRAC -mCherry and Cas9-RNPs targeting the AAVS1 and TRAC loci. (K) Proportion of transgene expressing cells co-transfected with nanoplasmid donors AAVS1 -mNG and TRAC -mCherry and Cas9-RNPs targeting the AAVS1 and TRAC loci that express mNG (green), mCherry (red), or both (blue). Circles represent individual donors; bars represent median values with range ( n = 4). This experiment was performed twice. *, P < 0.05; **, P < 0.01 in RM one-way ANOVA with Geisser–Greenhouse correction.

Article Snippet: Primary human CD8 + and CD4 + T cells were isolated by positive selection from buffy coats using the StraightFrom Buffy Coat CD8 MicroBead Kit or CD4 MicroBead Kit, respectively, according to the manufacturer’s instructions (Miltenyi Biotec).

Techniques: Gene Knock-In, Knock-In, Construct, Expressing, Electroporation, Cell Recovery, Transfection

Journal: The Journal of Experimental Medicine

Article Title: High-efficiency nonviral CRISPR/Cas9-mediated gene editing of human T cells using plasmid donor DNA

doi: 10.1084/jem.20211530

Figure Lengend Snippet: Multiplexed gene knock-in in human T cells. (A–C) Diagrams of pUC57 plasmid knock-in constructs are provided on the top. Representative contour plots (left) and bar graphs (right) showing the frequency of CD8 + T cells expressing mNG (A) 10 d after electroporation with a pUC57 plasmid TRAC -mNG donor template and Cas9-RNPs targeting the TRAC locus ( n = 3), mCherry (B) 10 d after electroporation with a pUC57 plasmid TRAC -mCherry donor template and Cas9-RNPs targeting the TRAC locus ( n = 3), or either mNG or mCherry (C) 10 d after electroporation with two pUC57 plasmid donor templates ( TRAC -mNG and TRAC -mCherry) and Cas9-RNPs targeting the TRAC locus ( n = 3). Graph on the right for C indicates proportion of transgene expressing cells that express mNG (green), mCherry (red), or both (blue). Circles represent individual donors; bars represent median values with range. This experiment was performed three times. (D) Diagrams of pUC57 plasmids used in dual targeting study, RAB11A -YFP and TRAC -mCherry. (E and F) Representative contour plot showing the frequency of CD8 + T cells expressing YFP, mCherry or both (E) and bar graphs (F) showing knock-in efficiency, cell viability, and total cell recovery of CD8 + T cells 10 d after electroporation with pUC57 donors RAB11A -YFP and TRAC -mCherry and Cas9-RNPs targeting the RAB11A and TRAC loci. (G) Proportion of transgene expressing cells co-transfected with pUC57 donor templates RAB11A -YFP and TRAC -mCherry and Cas9-RNPs targeting the RAB11A and TRAC loci that express YFP (green), mCherry (red), or both (blue). Circles represent individual donors; bars represent median values with range ( n = 4). This experiment was performed three times. *, P < 0.05; **, P < 0.01 in RM one-way ANOVA with Geisser–Greenhouse correction.

Article Snippet: Primary human CD8 + and CD4 + T cells were isolated by positive selection from buffy coats using the StraightFrom Buffy Coat CD8 MicroBead Kit or CD4 MicroBead Kit, respectively, according to the manufacturer’s instructions (Miltenyi Biotec).

Techniques: Gene Knock-In, Plasmid Preparation, Knock-In, Construct, Expressing, Electroporation, Cell Recovery, Transfection

Journal: The Journal of Experimental Medicine

Article Title: High-efficiency nonviral CRISPR/Cas9-mediated gene editing of human T cells using plasmid donor DNA

doi: 10.1084/jem.20211530

Figure Lengend Snippet: Nonviral CRISPR gene editing with large payloads. (A) Diagram of nanoplasmid knock-in constructs TRAC _NotchICD_mNG, TRAC_ NotchICD_1G4, and TRAC _THEMIS_1G4. (B, D, and F) Representative contour plots showing the frequency of CD8 + T cells expressing mNG (B) or 1G4 TCR (D and F) 5 d after electroporation of a NotchICD_mNG (B), NotchICD_1G4 (D), or THEMIS_1G4 (F) nanoplasmid donor template together with Cas9-RNP targeting the TRAC locus. (C, E, and G) Bar graphs showing the frequency of CD8 + T cells expressing mNG (C) or 1G4 TCR (E and G) and cell viability 5 d after electroporation of a NotchICD_mNG (C), NotchICD_1G4 (E), or THEMIS_1G4 (G) nanoplasmid donor template together with Cas9-RNP targeting the TRAC locus. Circles represent individual donors, and bars represent median values with range ( n = 3). This experiment was performed three times. *, P < 0.05; **, P < 0.01 in paired t test.

Article Snippet: Primary human CD8 + and CD4 + T cells were isolated by positive selection from buffy coats using the StraightFrom Buffy Coat CD8 MicroBead Kit or CD4 MicroBead Kit, respectively, according to the manufacturer’s instructions (Miltenyi Biotec).

Techniques: CRISPR, Knock-In, Construct, Expressing, Electroporation

Journal: Leukemia

Article Title: Spred1 deficit promotes treatment resistance and transformation of chronic phase CML

doi: 10.1038/s41375-021-01423-x

Figure Lengend Snippet: A Expression of SPRED1 in BM CD34+ cells from patients with BC CML and CP CML by Q-RT-PCR (n=8 samples for BC CML and n=12 samples for CP CML) and western blot and in BM by immunohistochemistry staining (one of three independent experiments with similar results was shown) (left), and expression of miR-126 in CD34+ and CD34+CD38− cells from BC CML (n=6 samples) and CP CML (n=10 samples) patients by Q-RT-PCR (right). B SPRED1 mRNA expression by Q-RT-PCR and protein expression by western blot, miR-126 levels by Q-RT-PCR, cell cycling by Ki-67 and DAPi staining (top) or by cell trace violet staining (bottom) followed by flow cytometry analysis in CML CD34+ cells transduced with SPRED1 siRNA to knock-down (KD) SPRED1 or with a non-targeting control siRNA (Ctrl). UND: undivided cells, G0; DIV: division. C Representative colonies and quantification of colony forming cells (CFC) in CML CD34+ (left) and CD34+CD38− (right) cells transduced with Spred1 siRNA to KD SPRED1 or with ctrl siRNA (n=3). Results shown represent mean ± SEM. Significance values: *, p<0.05; **, p<0.01; ***, p<0.001.

Article Snippet: Human CD34 + cells were selected using the indirect CD34 microbead kit (Miltenyi Biotec, San Diego, CA) and CD34 + CD38 − cells were sorted after staining with human antibodies against CD34 and CD38 ( Supplementary Table 1 ) or selected using CD34+CD38- cell isolation kit (Miltenyi Biotec, San Diego, CA) according to the manufacturer’s protocol.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunohistochemistry, Staining, Flow Cytometry, Transduction, Knockdown, Control

Journal: Frontiers in Immunology

Article Title: IgG-based B7-H3xCD3 bispecific antibody for treatment of pancreatic, hepatic and gastric cancer

doi: 10.3389/fimmu.2023.1163136

Figure Lengend Snippet: Induction of T cell activation against gastrointestinal cancer cell lines by CC-3. Monocyte-depleted PBMC of healthy donors were incubated with the indicated tumor cell lines (E:T 4:1) in the presence or absence of CC-3 or MOPCxCD3. If not stated otherwise, all constructs were used at 1 nM. (A) Activation of CD4+ and CD8+ T cells was determined by flow cytometric analysis for CD69 expression after 24 hours. Combined data obtained with PBMC of three independent donors are shown. (B) Degranulation of CD4+ and CD8+ T cells was determined by expression of CD107a after 4 h. Combined data obtained with PBMC of four independent donors are shown. (C) IL-2 and (D) IFNγ levels in culture supernatants were measured after 24 h using LEGENDplex assays. Combined data obtained with PBMC of four independent donors are shown. E:T, effector to target; PBMC, peripheral blood mononuclear cell.

Article Snippet: Where indicated, T cells within PBMC were isolated by using either Pan T cell Isolation Kit, human CD4 Micro Beads or human CD8 Micro Beads (Miltenyi Biotec).

Techniques: Activation Assay, Incubation, Construct, Expressing

Journal: Frontiers in Immunology

Article Title: IgG-based B7-H3xCD3 bispecific antibody for treatment of pancreatic, hepatic and gastric cancer

doi: 10.3389/fimmu.2023.1163136

Figure Lengend Snippet: Induction of T cell proliferation and memory T cell populations by CC-3. (A) Monocyte-depleted PBMC of healthy donors (n=4) were labelled with CellTrace™ Violet cell dye and incubated with or without MOPCxCD3 or CC-3 (1 nM each) in the presence of PANC-1, Hep3B, or NCI-N87 cells (E:T 4:1). After 72 h, PBMC were reexposed to fresh target cells and the respective treatment for additional 72 h. On day 6, proliferation was determined by flow cytometry. (B, C) PBMC of healthy donors (n=5) were incubated with or without MOPCxCD3 or CC-3 (1 nM each) in the presence of PANC-1, Hep3B, or NCI-N87 cells (E:T 1:1). After 72 h, cells were reexposed to fresh target cells and the respective treatment. On day 6, subpopulations of CD4 + and CD8 + T cells were determined by flow cytometric analysis. Effector T cells were defined as CD62L - CD45ro - , naive T cells as CD62L + CD45ro - , effector memory T cells as CD62L - CD45ro + and central memory T cells as CD62L + CD45ro + . (B) representative t-distributed stochastic neighbor embedding (tSNE) plots and (C) pooled data are shown. E:T, effector to target. PBMC, peripheral blood mononuclear cell.

Article Snippet: Where indicated, T cells within PBMC were isolated by using either Pan T cell Isolation Kit, human CD4 Micro Beads or human CD8 Micro Beads (Miltenyi Biotec).

Techniques: Incubation, Flow Cytometry

Journal: Molecular cell

Article Title: Mitophagy Controls the Activities of Tumor Suppressor p53 to Regulate Hepatic Cancer Stem Cells

doi: 10.1016/j.molcel.2017.09.022

Figure Lengend Snippet: (a) HepG2 with or without various treatments for 24 hours and stable HepG2 cells that expressed a control shRNA (sh-Ctrl) or the Atg5 shRNA (sh-Atg5) were subjected to flow cytometry analysis for CD133+ cells. Results represent the mean ± SEM of three independent experiments. None, no treatment. (B) HepG2 cells with the treatments shown in (A) were lysed for immunoblot analysis. LC3-I, non-lipidated LC3; LC3-II, lipidated LC3. The β-actin protein was also analyzed to serve as the loading control. (C) Sphere-formation assay of CD133+ and CD133− HepG2 cells. The panels shown to the left are representative results of spheres formed by CD133+ and CD133− HepG2 cells with and without stable ATG5 knockdown. Scale bar=200 μm. The histogram shown to the right indicated the number of spheres larger than 100 μm in diameter when 500 CD133+ cells were seeded. The results represent the mean ± SEM of three independent experiments. (D) HepG2 cells with various treatments for 24 hours were incubated with MicroBeads (Miltenyi Biotec) for the isolation of CD133+ cells, which were then analyzed for their sphere-forming ability. 500 cells were seeded for the assay. Also see Figure S1.

Article Snippet: Sphere-formation assay The Human CD133 MicroBead Kit (Miltenyi Biotec) was used to isolate CD133 + cells from HepG2, Hep3B and Huh7 cells.

Techniques: Control, shRNA, Flow Cytometry, Western Blot, Tube Formation Assay, Knockdown, Incubation, Isolation

Journal: Molecular cell

Article Title: Mitophagy Controls the Activities of Tumor Suppressor p53 to Regulate Hepatic Cancer Stem Cells

doi: 10.1016/j.molcel.2017.09.022

Figure Lengend Snippet: (A) Hep3B and Huh7 cells with various treatments for 24 hours were subjected to flow cytometry analysis for CD133+ cells. (B) HepG2 and Huh7 cells were transfected with the p53-expressing plasmid for two days, treated with 3-MA or rapamycin for another 24 hours and then subjected to flow cytometry analysis for CD133+ cells. (C) HepG2 cells transfected with the control siRNA (si-Ctrl) or the p53 siRNA (si-p53) for two days or treated with PFTα or DMSO for one day were analyzed for their CD133+ cells by flow cytometry (top panel) or sphere-forming ability of their CD133+ cells (bottom panel). The results shown in (A), (B) and (C) represent the mean ± SEM of three independent experiments. (D) HepG2 cells treated with DMSO or PFTα for one day or with siRNA for two days were lysed for immunoblot analysis. (E) Stable HepG2 cells that expressed control shRNA (sh-Ctrl), sh-Atg5, or both sh-Atg5 and sh-p53 were lysed for immunoblot analysis. (F) Cells mentioned in (E) were used for the sphere-formation assay. Also see Figure S2.

Article Snippet: Sphere-formation assay The Human CD133 MicroBead Kit (Miltenyi Biotec) was used to isolate CD133 + cells from HepG2, Hep3B and Huh7 cells.

Techniques: Flow Cytometry, Transfection, Expressing, Plasmid Preparation, Control, Western Blot, shRNA, Tube Formation Assay

Journal: Molecular cell

Article Title: Mitophagy Controls the Activities of Tumor Suppressor p53 to Regulate Hepatic Cancer Stem Cells

doi: 10.1016/j.molcel.2017.09.022

Figure Lengend Snippet: (A) HepG2 cells with various treatments for 24 hours or stably expressing the control shRNA or the Atg5 shRNA were lysed for immunoblot analysis. (B) Immunoblot analysis of HepG2 cells transfected with either the control vector or the expression vector for various p53 proteins. Cells were lysed two days after DNA transfection for immunoblot analysis. None, control cells with no DNA transfection. (C) The experiments were conducted the same way as in (B), with the exception that Hep3B cells were used for the expression studies. (D) Hep3B or HepG2 cells were transfected with various p53-expressing plasmids or the control vector as indicated for two days followed by flow cytometry analysis for CD133+ cells. (E) HepG2 cells were transfected with the p53-expressing plasmids for two days, and CD133+ cells were then isolated for the sphere-formation assay. (F) The experiments were conducted the same way as in (E), with the exception that Hep3B cells were used for the studies. The results in (D–F) represented the mean ± SEM of three independent experiments. Also see Figure S3.

Article Snippet: Sphere-formation assay The Human CD133 MicroBead Kit (Miltenyi Biotec) was used to isolate CD133 + cells from HepG2, Hep3B and Huh7 cells.

Techniques: Stable Transfection, Expressing, Control, shRNA, Western Blot, Transfection, Plasmid Preparation, Flow Cytometry, Isolation, Tube Formation Assay

Journal: Molecular cell

Article Title: Mitophagy Controls the Activities of Tumor Suppressor p53 to Regulate Hepatic Cancer Stem Cells

doi: 10.1016/j.molcel.2017.09.022

Figure Lengend Snippet: (A) Top panel, HepG2 cells were treated with DMSO, Mdivi-1 or CCCP for one day and then subjected to flow cytometry analysis for CD133+ cells; bottom panel, CD133+ HepG2 cells were isolated and treated with Mdivi-1 or CCCP for two days and then analyzed for their sphere-forming ability. (B) HepG2 cells without treatment or with the treatment of DMSO, CCCP or Mdivi-1 for one day were lysed for immunoblot analysis. Cells were also subjected to subcellular fractionation for the isolation of mitochondria, cytosol, and nuclei for immunoblot analysis. Tom20, β-actin and lamin B1 were used as the loading controls for mitochondria, cytosol and nucleus, respectively, to ensure equal amount of proteins were loaded on the gel. (C) Confocal microscopy for the analysis of the subcellular localization of p53(pS392) in HepG2 cells treated with DMSO, Mdivi-1 or CCCP. TOM20 was used as the marker for mitochondria. The areas boxed are enlarged at the bottom. Scale bar, 10 μm. (D) The results shown in (C) were quantified with a Leica TCS SP8 fluorescent confocal microscope. The results indicated the percentages of p53(pS392) that colocalized with TOM20. The results represent the mean ± SEM of at least 30 cells that were analyzed. See also Figure S5.

Article Snippet: Sphere-formation assay The Human CD133 MicroBead Kit (Miltenyi Biotec) was used to isolate CD133 + cells from HepG2, Hep3B and Huh7 cells.

Techniques: Flow Cytometry, Isolation, Western Blot, Fractionation, Confocal Microscopy, Marker, Microscopy

Journal: Molecular cell

Article Title: Mitophagy Controls the Activities of Tumor Suppressor p53 to Regulate Hepatic Cancer Stem Cells

doi: 10.1016/j.molcel.2017.09.022

Figure Lengend Snippet: (A) Effects of PINK1 knockdown on CD133+ HepG2 cells (top panel), their sphere-forming ability (middle panel) and their effects on the Nanog promoter using the Nanog-luc1 reporter (bottom panel). HepG2 cells transfected with either the control siRNA or the PINK1 siRNA for two days were analyzed. In the bottom panel, HepG2 cells were also transfected with the Nanog-luc1 reporter (see Figure 3B) for the analysis of luciferase activity. The luciferase activity of cells without the transfection of siRNA was arbitrarily defined as 1. The results represented the mean ± SEM of three independent experiments. (B) Effects of PINK1 over-expression on CD133+ HepG2 cells (top panel), their sphere-forming ability (middle panel) and their effects on the Nanog promoter (bottom panel). The experiments were conducted the same way as in (A), except that instead of using siRNA, cells were transfected with either the control vector or the PINK1-expressing plasmid. (C) Immunoblot analysis of HepG2 cells with PINK1 knockdown (left panels) or PINK1 over-expression (right panels) were lysed for immunoblot analysis. Total cell lysates as well as the nuclear lysates (bottom two panels) were analyzed. (D) PINK1 in HepG2, Hep3B or Huh7 cells was immunoprecipitated with a control antibody (−) or the anti-PINK1 antibody (+) and then incubated with GST-p53 in the presence of ATP. The GST-p53 phosphorylated at S392 was analyzed using the anti-p53 antibody that recognized phosphoserine-392. GST-p53 added in the reaction and PINK1 immunoprecipitated were also analyzed by immunoblot (bottom two panels). Numbers to the left of the top panel indicate protein molecular weight markers. (E) GST-p53 was mixed with GST-PINK1 or GST and incubated in the presence of ATP. The phosphorylation of p53 at S392 was then analyzed with the antibody that recognized phosphoserine-392. GST-p53, GST-PINK1 and GST used for the reaction was also analyzed by anti-p53, anti-PINK1 and anti-GST antibodies, respectively (bottom three panels). Numbers to the left indicate protein molecular weight markers. (F) Co-immunoprecipitation of p53 and p53(pS392) with PINK1. HepG2 cells were lysed and immunoprecipitated using the anti-PINK1 antibody or the control antibody followed by immunoblot analysis for p53, p53(pS392) and PINK1. (G) Co-immunoprecipitation of p53 and p53(pS392) with PINK1 using the anti-PINK1 antibody in different subcellular fractions (top 3 panels). β-actin, lamin B1 and Tom20 were used as the markers for cytosolic (C), nuclear (N) and mitochondrial (M) fractions. Equal amounts of p53 were used for the co-immunoprecipitation experiment (bottom 2 panels). See also Figure S6.

Article Snippet: Sphere-formation assay The Human CD133 MicroBead Kit (Miltenyi Biotec) was used to isolate CD133 + cells from HepG2, Hep3B and Huh7 cells.

Techniques: Knockdown, Transfection, Control, Luciferase, Activity Assay, Over Expression, Plasmid Preparation, Expressing, Western Blot, Immunoprecipitation, Incubation, Molecular Weight, Phospho-proteomics

Journal: Molecular cell

Article Title: Mitophagy Controls the Activities of Tumor Suppressor p53 to Regulate Hepatic Cancer Stem Cells

doi: 10.1016/j.molcel.2017.09.022

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Sphere-formation assay The Human CD133 MicroBead Kit (Miltenyi Biotec) was used to isolate CD133 + cells from HepG2, Hep3B and Huh7 cells.

Techniques: Virus, Recombinant, Plasmid Preparation, Bicinchoninic Acid Protein Assay, Mouse Assay, Mutagenesis, Software, Imaging, Extraction, Isolation, DNA Labeling

Journal: Nature

Article Title: TGFβ links EBV to multisystem inflammatory syndrome in children

doi: 10.1038/s41586-025-08697-6

Figure Lengend Snippet: a , b , GSEA using a previously defined TGFβ gene set applied to T cells ( a ) and the gene set ‘Li M200 antigen processing and presentation’ applied to monocyte clusters ( b ) depicted as both UMAP (left) and a dot plot (right). c , Schematic overview of T cell reactivation assays. d , Frequencies of overall activated (CD69 + ) and antigen-specific reactivated (CD137 + CD69 + and CD154 + CD69 + ) CD4 + or CD8 + memory T cells (T mem , CD45RO + ) from patients with MIS-C during the acute phase and at follow-up after symptoms resolved ( n = 8 patients and n = 5 different viral peptides). e , f , Frequencies of overall activated and antigen-specific reactivated cells of CD4 + and CD8 + memory T cells (T mem ; CD45RO + ) from healthy donors ( n = 6) treated with serum from patients with MIS-C ( e ; n = 7) or patients with severe COVID-19 ( f ; n = 5) with or without anti-TGFβ. Samples that were obtained more than 24 h after the start of treatment are colour-coded in yellow. Unpaired ( a , b ) or paired ( d – f ) two-tailed Mann–Whitney U -tests.

Article Snippet: After stimulation, cells were stained with TotalSeq anti-human Hashtags as previously mentioned, followed by CD154 MACS enrichment according to the manufacturer’s protocol (CD154 MicroBead Kit, human; Miltenyi Biotec).

Techniques: Two Tailed Test, MANN-WHITNEY

Journal: Nature

Article Title: TGFβ links EBV to multisystem inflammatory syndrome in children

doi: 10.1038/s41586-025-08697-6

Figure Lengend Snippet: a , Gating strategy used in flow cytometry of the T cell reactivity assays depicted in Fig. . Cells were identified by size and granularity in a FSC-vs SSC plot, followed by doublet exclusion in an FSC-A vs. FSC-H plot. Dump + (DAPI, CD14 and CD19) + cells were also excluded. As CD3 is downregulated after T cell activation (SEB plot in second row), the gate was extended to include CD3 low CD45RO + cells. CD4 + epitope-specific T cells were identified as CD69 + CD154 + and CD8 + epitope specific T cells were identified as CD69 + CD137 + or as CD69 + CD154 + . SEB was used as a positive control for correct gating. b , Cell counts for CD69 + or CD69 + and CD154 + or CD137 + memory T cells from Fig. . c , Gating strategy used in flow cytometry of the T cell reactivity assays depicted in d. d , Frequencies of overall activated and antigen-specific reactivated cells of CD4 + and CD8 + memory T cells from six children with a confirmed infection with SARS-CoV-2 during the acute phase and follow-up upon after resolution of symptoms. e , Frequencies of overall activated and antigen-specific reactivated cells of CD4 + and CD8 + memory T cells from healthy donors ( n = 6) treated with 50 ng ml −1 TGFβ1. f , Frequencies of TCRVβ21.3 + on total T cells were quantified by Flow cytometry over time after treatment start with IVIG and methylprednisolone. Horizontal lines indicate normal range (0.9-4.9% for CD8 + T cells; 1.5-4-7% for CD4 + T cells) of TCRVβ21.3 + T cells ( n = 25, children with MIS-C). g , Significantly regulated TRBV determined by TCR sequencing of activated T cells. Dots indicate the frequency of specific TRBV in each sample relative to all TCRs sequenced. h , Frequencies of TRAV gene associated to TRBV11-2 + T cells not depicted in Fig. . i , HLA-class I haplotyping and ( j-k ) HLA-class-II haplotyping of our MIS-C cohort ( n = 20 patients and n = 10 healthy controls including the 4 children used as a control for the scRNAseq experiments). Additionally, HLA-haplotyping from a previously published MIS-C cohort ( n = 7 patients and 9 controls) was included. l-m , Sorting strategy for Fig. . P -values for ( b + d-e + h ) were determined by paired two-tailed Mann-Whitney- U -tests.

Article Snippet: After stimulation, cells were stained with TotalSeq anti-human Hashtags as previously mentioned, followed by CD154 MACS enrichment according to the manufacturer’s protocol (CD154 MicroBead Kit, human; Miltenyi Biotec).

Techniques: Flow Cytometry, Activation Assay, Positive Control, Infection, Sequencing, Control, Two Tailed Test, MANN-WHITNEY

Journal: Nature

Article Title: TGFβ links EBV to multisystem inflammatory syndrome in children

doi: 10.1038/s41586-025-08697-6

Figure Lengend Snippet: a , Schematic showing generation of virus-specific TCR libraries and comparison of virus-specific TCRs with MIS-C-specific TCRs. scTCR-seq, single-cell TCR sequencing. b , UMAP of 22,344 virus-specific T cells from donors restimulated with EBV ( n = 5), CMV ( n = 5), SARS-CoV-2 ( n = 3) or measles ( n = 3) peptides, representing 18,010 sequenced TCRβ chains and 15,496 full TCRs. AdV-specific T cells were TCR-sequenced. Virus-specificities are colour-coded. c , TRVB11-2 + T cells superimposed on the UMAP in b . d , Gene expression superimposed on the UMAP of antigen-specific T cells, showing that most TRVB11-2 + T cells have a CD4 or CD8 cytotoxic phenotype (low: ICOS ; high: PRF1 , GZMB , LAMP1 ). e , TCR repertoires of EBV ( n = 5), CMV ( n = 5), SARS-CoV-2 ( n = 3), measles ( n = 3) and AdV ( n = 1) virus-specific T cells from healthy donors, analysed by ARTE . Heat map showing distribution of TRAV gene expression associated with TRBV11-2 -positive T cells in virus-specific and MIS-C T cells ( n = 11) T cells, compared with 6 wpi no MIS-C ( n = 4) and paediatric influenza ( n = 3) T cells. Unsupervised clustering was performed with the R package pheatmap. f , TCRVβ21.3 expression on memory T (T mem ) cells after stimulation with EBNA2 275–294 (left) or EBNA2 279–289 (right) peptides, analysed by ARTE. Frequencies of TCRVβ21.3 + in all CD4 + (top) and CD8 + (bottom) memory T cells and those with antigen-specific reactivation (CD154 + CD69 + ) from n = 7 donors. Flow cytometry gating is shown in Extended Data Fig. . Two-sided paired t -test.

Article Snippet: After stimulation, cells were stained with TotalSeq anti-human Hashtags as previously mentioned, followed by CD154 MACS enrichment according to the manufacturer’s protocol (CD154 MicroBead Kit, human; Miltenyi Biotec).

Techniques: Virus, Comparison, Sequencing, Gene Expression, Expressing, Flow Cytometry