cd44v6 pe  (R&D Systems)

Journal: Molecular Therapy. Nucleic Acids

Article Title: Single-cell analysis of the survival mechanisms of fratricidal CAR-T targeting of T cell malignancies

doi: 10.1016/j.omtn.2024.102225

Figure Lengend Snippet: Study design and clustering analysis of CAR-T scRNA-seq data (A) Fold change in the proliferation of nontransfected (NT) cells or CD44v6 CAR-T cells after 14 days of transduction compared with day 0. (B) Apoptosis of NT cells or CD44v6 CAR-T cells after 7 and 14 days of transduction. (C) Quantification of cytokines IFN-γ, TNF-α, IL-6, and IL-4 in the supernatant of NT or CD44v6 CAR-T cells after 14 days transfection via BD cytometric bead array. (D) Schematic of the study design for scRNA-seq and scTCR-seq. Peripheral blood mononuclear cells (PBMCs) were collected from two healthy donors, and T cells were sorted by CD3 + staining. A CAR of CD19, CD26, or CD44v6 was constructed; after 14 days scRNA-seq combined with scTCR-seq was performed using the 10× Genomics platform. (E) Bar charts show the fraction of cells (left) and number of cells (right) in each sample among cell subpopulations. (F) Dot plot showing scaled expression levels of typical marker genes for each cell subpopulation colored by average expression. Dot size represents the percentage of cells in each cell subpopulation with more than one read of the corresponding gene. (G) UMAP plot of pooled CAR + T cells collected from all conditions of six samples. Each point represents a single cell, colored according to cell subpopulation. Statistical significance: ∗ p < 0.05, ∗∗∗ p < 0.001.

Article Snippet: For the expression of CD19, CD26, and CD44v6 markers on the surface of the T cells, T cells were incubated with CD19-BV421 (BD Biosciences, cat. no. 562441), CD26-PE (BD Biosciences, cat. no. 555437), and CD44v6-PE (BD Biosciences, cat. no. 566803) antibodies for 30 min at 4°C in the dark.

Techniques: Transduction, Transfection, Staining, Construct, Expressing, Marker

Journal: Molecular Therapy. Nucleic Acids

Article Title: Single-cell analysis of the survival mechanisms of fratricidal CAR-T targeting of T cell malignancies

doi: 10.1016/j.omtn.2024.102225

Figure Lengend Snippet: The expression of the CAR-T target genes CD26 and CD44v6 in all cell subpopulations (A) The percentages of CD19-expressing (CD19 + ) cells (left), CD26-expressing (CD26 + ) cells (middle), and CD44v6-expressing (CD44v6 + ) cells (right) in untransfected T cells were analyzed by flow cytometry. (B) Percentage of CD26 + cells (top) and CD44-expressing (CD44 + ) cells (bottom) in all CAR + cell subpopulations from single-cell sequencing data. (C) Log-normalized expression of the CD26 and CD44 genes for 10 T cell subpopulations. (D) Dot plot for the expression of the CD26, CD44, and CAR genes in CAR + cells of two donor samples.

Article Snippet: For the expression of CD19, CD26, and CD44v6 markers on the surface of the T cells, T cells were incubated with CD19-BV421 (BD Biosciences, cat. no. 562441), CD26-PE (BD Biosciences, cat. no. 555437), and CD44v6-PE (BD Biosciences, cat. no. 566803) antibodies for 30 min at 4°C in the dark.

Techniques: Expressing, Flow Cytometry, Sequencing

Journal: Molecular Therapy. Nucleic Acids

Article Title: Single-cell analysis of the survival mechanisms of fratricidal CAR-T targeting of T cell malignancies

doi: 10.1016/j.omtn.2024.102225

Figure Lengend Snippet: Key subpopulations and their clonal expansion analyses of CAR + cells in CD26 and CD44v6 CAR-T (A) Bar charts show the percentage of each cell subpopulation among all CAR-expressing (CAR + ) cells in each sample. (B) Relative contents of all CAR + cell subpopulations (compared with CD19 CAR-T sample of the same individual) in all samples. Red font represents increased cell content in fratricidal CAR-T cells, and blue represents decreased. (C) Heatmap shows the CAR-T sample preference of various cell subpopulations with detected TCRs estimated by the STARTRAC-dist index of STARTRAC (see ), in which R o/e is the ratio of observed cell number over the expected cell number of a given combination of T cell subpopulation and sample. (D) Clonal expansion levels of different T cells quantified by STARTRAC-expa for each CAR-T cell subpopulation. (E) Distribution of proliferation score (x axis) vs. the clonal expansion index (y axis, STARTRAC-expa index). (F) TCR diversity of each cell subpopulation (Inv.Simpson index) among the three CAR-T types. Two-tailed paired t test; ∗∗∗∗ p < 0.0001.

Article Snippet: For the expression of CD19, CD26, and CD44v6 markers on the surface of the T cells, T cells were incubated with CD19-BV421 (BD Biosciences, cat. no. 562441), CD26-PE (BD Biosciences, cat. no. 555437), and CD44v6-PE (BD Biosciences, cat. no. 566803) antibodies for 30 min at 4°C in the dark.

Techniques: Expressing, Two Tailed Test

Journal: Molecular Therapy. Nucleic Acids

Article Title: Single-cell analysis of the survival mechanisms of fratricidal CAR-T targeting of T cell malignancies

doi: 10.1016/j.omtn.2024.102225

Figure Lengend Snippet: Differential expression analysis between fratricidal CAR-T cells and CD19 CAR-T cells (A) Dot plots of the expression levels of differentially expressed cytokine genes (cytokine DEGs) in CD4 + central memory T cells, CD8 + cytotoxic T cells, exhausted T cells, helper T cells, and MT + effector T cells of two donor samples. (B) Volcano plot of up- and downregulated genes in CD26 and CD44v6 CAR-T cells compared with CD19 CAR-T cells in CD4 + central memory T cells. Genes with |log2(FoldChange)| ≥ 0.25 in both comparison groups (CD26 vs. CD19 and CD44v6 vs. CD19) are highlighted. (C) Heatmap of differentially expressed immune-related genes, including immunoinhibitor, immunostimulator, and MHC molecules, in CD4 + central memory T cells. Each column represents a cell, while each row indicates a gene. (D) Significantly (false discovery rate [FDR] ≤ 0.05) enriched Gene Ontology biological process (BP) of consistently up- or downregulated genes from CD26 and CD44v6 CAR-T cells compared with CD19 CAR-T cells in CD4 + central memory T cells. Dot color indicates the statistical significance of the enrichment (−log FDR), and dot size represents the gene ratio enriched in each term. Red terms, T cell and immune-related terms.

Article Snippet: For the expression of CD19, CD26, and CD44v6 markers on the surface of the T cells, T cells were incubated with CD19-BV421 (BD Biosciences, cat. no. 562441), CD26-PE (BD Biosciences, cat. no. 555437), and CD44v6-PE (BD Biosciences, cat. no. 566803) antibodies for 30 min at 4°C in the dark.

Techniques: Expressing, Comparison

Journal: Molecular Therapy. Nucleic Acids

Article Title: Single-cell analysis of the survival mechanisms of fratricidal CAR-T targeting of T cell malignancies

doi: 10.1016/j.omtn.2024.102225

Figure Lengend Snippet: Differential expression and regulatory analysis of exhausted T cells in three CAR-T types (A) Volcano plot of up- and downregulated genes in CD26 and CD44v6 CAR-T cells compared with CD19 CAR-T in exhausted T cells. Genes with |log2(FoldChange)| ≥ 0.25 in both comparison groups (CD26 vs. CD19 and CD44v6 vs. CD19) are highlighted. (B and C) Significantly enriched (FDR ≤ 0.05) KEGG pathways (B) and GO biological processes (C) of DEGs from CD26 and CD44v6 CAR-Ts compared with CD19 CAR-Ts in exhausted T cells. Dot color indicates the statistical significance of the enrichment (−log FDR), and dot size represents the gene count enriched in each term. (D) Heatmap of the normalized regulon activity ( Z score, see ) derived from a generalized linear model of the difference between the three CAR-T types in exhausted T cells. Each column represents a cell, while each row indicates a regulon. A regulon name such as “RUNX3 (67g)” means a gene-regulatory network consisting of TF RUNX3 with its 67 high-confidence target genes. A regulon name such as “RUNX3_extended (76g)” indicates a gene-regulatory network consisting of TF RUNX3 with all 76 target genes. (E) Dot plot showing the scaled average expression of TFs in (D) for the three CAR-T-cell types. Red genes represent upregulation in fratricidal CAR-T cells, while blue genes represent downregulation. (F) Based on the regulons in (D), regulatory networks of key regulators on immune-related genes were constructed. The thickness of the edge represents the confidence score of the corresponding TF-regulated target gene.

Article Snippet: For the expression of CD19, CD26, and CD44v6 markers on the surface of the T cells, T cells were incubated with CD19-BV421 (BD Biosciences, cat. no. 562441), CD26-PE (BD Biosciences, cat. no. 555437), and CD44v6-PE (BD Biosciences, cat. no. 566803) antibodies for 30 min at 4°C in the dark.

Techniques: Expressing, Comparison, Activity Assay, Derivative Assay, Construct

Journal: Molecular Therapy. Nucleic Acids

Article Title: Single-cell analysis of the survival mechanisms of fratricidal CAR-T targeting of T cell malignancies

doi: 10.1016/j.omtn.2024.102225

Figure Lengend Snippet: Function changes of CD44v6 CAR-T cells when the exhaustion was suppressed (A) Experimental design of CD44v6 CAR-T cells treated with the Tim3 inhibitor sabatolimab or LAG3 inhibitor relatlimab. (B) Tim3 (top) and LAG3 (bottom) expression levels in CD44v6 CAR-T cells treated with the Tim3 inhibitor sabatolimab or LAG3 inhibitor relatlimab at 10 and 20 μM for 7 and 14 days. Representative flow cytometry is shown on the left, and Tim3 or LAG3 expression analysis is shown on the middle and right. (C) Proliferation levels of CD44v6 CAR-T cells treated with Tim3 inhibitor (left) or LAG3 inhibitor (right). (D) Apoptosis of CD44v6 CAR-T cells treated with Tim3 inhibitor (left) or LAG3 inhibitor (right). (E) Specific cytotoxicity to tumor of CD44v6 CAR T cells treated with Tim3 and LAG3 inhibitors for 14 days. These experiments were repeated for five healthy donors. Data are depicted as the mean ± SD. Significance was determined by two-way ANOVA. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant.

Article Snippet: For the expression of CD19, CD26, and CD44v6 markers on the surface of the T cells, T cells were incubated with CD19-BV421 (BD Biosciences, cat. no. 562441), CD26-PE (BD Biosciences, cat. no. 555437), and CD44v6-PE (BD Biosciences, cat. no. 566803) antibodies for 30 min at 4°C in the dark.

Techniques: Expressing, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: CD44v6 specific CAR-NK cells for targeted immunotherapy of head and neck squamous cell carcinoma

doi: 10.3389/fimmu.2023.1290488

Figure Lengend Snippet: Optimization of the anti-CD44v6 retroviral vector CAR delivery and expression in primary NK cells. (A) Schematic representation of the anti-CD44v6 CAR construct used to generate CAR-NK cells. The anti-CD44v6 scFv (aCD44v6 scFv) derives from the BIWA8 clone of the anti-CD44v6 humanized antibody bivatuzumab. A human IgG1 hinge domain (huIgG1 hinge) links the scFv to a CD8 transmembrane domain (CD8a TMD), CD28 costimulatory domain (CD28) and CD3ζ signaling domain (CD3ζ). The genes for the enhanced GFP (EGFP) reporter and the CAR are connected through a P2A self-cleaving peptide sequence. The constitutive expression of the CAR gene in primary NK is controlled by the cytomegalovirus (CMV) promoter. The expression cassette is cloned into a gamma retroviral vector (gRV). (B) Example of gating strategy and comparison between different MOIs of the gamma retroviral vectors produced in HEK293-T cells and used to transduce primary NK cells. The two-parameter dot plots represent EGFP+, EGFP+CAR+ and CAR+ expression in transduced primary NKs with gRV pseudotyped with BaEV on day 10 post transduction. CAR surface expression was detected by staining with an APC IgG1 monoclonal antibody. (C) NK-cell transduction and percentage of total transduced cells (EGFP+, EGFP+CAR+, CAR+) at days 3, 7, 10 and 14 post transductions with gRV pseudotyped with BaEV, RD114 and GaLV. Unconcentrated viral vectors (UNC) and three MOIs of 0.5, 1 and 5 concentrated viral vectors were used to transduce NK cells. Expanded NK cells (EXP pNKs) and the process control NK cells (PC pNKs) serve as controls. Data of five independent experiments using five different healthy NK cells donors is presented as mean and standard deviation.

Article Snippet: Staining was carried out according to the manufacturer’s instructions using the following antibodies: CD44v6-PE (Becton, Dickson and Company, New Jersey, USA); PD-L1-PE-Vio770; IgG1-APC; CD8-FITC; CD4-PE; CD19-PE-Vio770; CD16-PE-Vio 615; CD14-APC, CD3-VioBlue; CD45-VioGreen; NKG2A-PE; NKp44-Vio Bright B515; NKG2D-APC; NKp46-PE-Vio 615; CD16-VioGreen; CD56-APC-Vio 770; PD-1-PE-Vio770; CD69-PerCP-Vio 700; DNAM-1-Vio Bright R720; TIGIT-PE.

Techniques: Retroviral, Plasmid Preparation, Expressing, Construct, Sequencing, Clone Assay, Comparison, Produced, Transduction, Staining, Control, Standard Deviation

Journal: Frontiers in Immunology

Article Title: CD44v6 specific CAR-NK cells for targeted immunotherapy of head and neck squamous cell carcinoma

doi: 10.3389/fimmu.2023.1290488

Figure Lengend Snippet: Killing efficacy of anti-CD44v6 CAR-NK cells against different HNSCC cell lines after 4, 6, 8 and 24 h of co-culture. (A) Schematic representation of the kinetic killing assay in which target cells genetically modified to express firefly luciferase (F.luc) are co-cultured with effector NK cells. Killing efficacy is monitored by recording living cells through the use of D-luciferin as substrate. This substrate is reduced to oxyluciferin by living cells. The bioluminescent signal is quantified and normalized to indicate cell killing. (B–D) CD44v6 positive UT-SCC-14-F.luc; UT-SCC-42B-F.luc and SCC-25-F.luc cells are set in co-culture with EXP pNKs; PC pNKs and CD44v6 CAR-NKs at 2.5:1 and 1:1 effector to target ratios. Killing efficacy is quantified after 4, 6 and 8 h of co-culture (n=5 donors). (E–G) Killing efficacy against the same target cell lines was checked after 24 h using 2.5:1, 1:1 and 0.1:1 effector to target ratios (n=3 donors). Data is presented as mean and standard deviation of 5 or 3 independent experiments. Descriptive statistics were calculated using two-way ANOVA and Turkey’s multiple comparison test (* p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).

Article Snippet: Staining was carried out according to the manufacturer’s instructions using the following antibodies: CD44v6-PE (Becton, Dickson and Company, New Jersey, USA); PD-L1-PE-Vio770; IgG1-APC; CD8-FITC; CD4-PE; CD19-PE-Vio770; CD16-PE-Vio 615; CD14-APC, CD3-VioBlue; CD45-VioGreen; NKG2A-PE; NKp44-Vio Bright B515; NKG2D-APC; NKp46-PE-Vio 615; CD16-VioGreen; CD56-APC-Vio 770; PD-1-PE-Vio770; CD69-PerCP-Vio 700; DNAM-1-Vio Bright R720; TIGIT-PE.

Techniques: Co-Culture Assay, Genetically Modified, Luciferase, Cell Culture, Standard Deviation, Comparison