Journal: bioRxiv

Article Title: Simultaneous, cell-intrinsic downregulation of PD-1 and TIGIT enhances the effector function of CD19-targeting CAR T cells and promotes an early-memory phenotype

doi: 10.1101/2020.11.07.372334

Figure Lengend Snippet: (a) Schematic representation of the lentiviral two-in-one vector carrying a CD19-CAR and shRNA expressing module. (b) PD-1 expression levels of CAR T cells with different shPD-1 candidates as determined by flow cytometry on day two after stimulation with γ-irradiated Nalm-6-GL cells. Gray denotes the isotype control. Data are the pooled mean ± SD from three independent experiments, each performed in triplicates. (c) Cell counts from the homeostatic expansion of LNGFR + CAR T cells with PD-1 downregulation candidates on days 3 and 6 after cell seeding. Data are the pooled mean ± SD from three independent experiments performed in triplicates. (d) The effects of hH1-, hU6-, and mU6-shPD1 on PD-1 expression was analyzed by flow cytometry two days after stimulation with γ-irradiated K562-CD19 cells. Data are the pooled mean ± SD from two independent experiments performed in triplicates. (e) Cell counts from the homeostatic expansion of CAR T cells with each Pol III promoter after LNGFR + isolation on days 3 and 6 after cell seeding. Data are the pooled mean ± SD from two independent experiments performed in triplicates. (f) CAR T cells were incubated with GFP-expressing Nalm-6-GL or Nalm-6-GL-PD-L1 cells at a 1:1, 0.3:1, and 0.1:1 effector: target (E:T) ratio. GFP intensity was measured every two hours using the IncuCyte S3 live-cell imaging system. The relative percentage of total integrated GFP intensity was calculated as [GFP intensity at each time point / GFP intensity at 0 h]*100. Representative mean ± SD from two independent experiments performed in triplicates. (g) CAR T cells were incubated with γ-irradiated K562-CD19 or K562-CD19-PD-L1 cells at a 1:3 E:T ratio and counted on day 7. Data are the pooled mean ± SD from two independent experiments performed in triplicates. (h) NSG mice were injected intravenously with 1×10 6 Nalm-6-GL-PD-L1 leukemia cells. 5 days later, 1×10 6 CAR T cells were injected intravenously. Tumor burden was monitored based on the bioluminescence intensity from the IVIS imaging system. Data are from n = 3 mock and n = 5 19BBz, 19GBBz, and 19PBBz mice, respectively, (i) PD-1 expression levels of CAR T cells from Nalm-6-GL-PD-L1-bearing mice at day 43. Gray denotes isotype control. Data are the mean ± SD from three mice per group. Statistical analysis was done by One-Way ANOVA for (b-f) and unpaired two-tailed t-test for (g and i). *p < 0.05, ** p < 0.01, *** = p < 0.001, **** = p < 0.0001, ns = not significant.

Article Snippet: Nalm-6-GL, or K562-CD19 cells were transduced to express human PD-L1 (Sino Biological; HG10084-UT cDNA subcloned into a lentiviral vector) to generate Nalm-6-GL-PD-L1 and K562-CD19-PD-L1 cells.

Techniques: Plasmid Preparation, shRNA, Expressing, Flow Cytometry, Irradiation, Isolation, Incubation, Live Cell Imaging, Injection, Imaging, Two Tailed Test

Journal: bioRxiv

Article Title: Simultaneous, cell-intrinsic downregulation of PD-1 and TIGIT enhances the effector function of CD19-targeting CAR T cells and promotes an early-memory phenotype

doi: 10.1101/2020.11.07.372334

Figure Lengend Snippet: Surface CD19, PD-L1, and CD80 expression levels of Nalm-6-GL-PD-L1-CD80, Nalm-6-GL-PD-L1, Nalm-6, K562-CD19-PD-L1, K562-CD19, and K562 cells was determined by flow cytometry.

Article Snippet: Nalm-6-GL, or K562-CD19 cells were transduced to express human PD-L1 (Sino Biological; HG10084-UT cDNA subcloned into a lentiviral vector) to generate Nalm-6-GL-PD-L1 and K562-CD19-PD-L1 cells.

Techniques: Expressing, Flow Cytometry

Journal: bioRxiv

Article Title: Simultaneous, cell-intrinsic downregulation of PD-1 and TIGIT enhances the effector function of CD19-targeting CAR T cells and promotes an early-memory phenotype

doi: 10.1101/2020.11.07.372334

Figure Lengend Snippet: (a) PD-1 expression level of CAR T cells with CD28 or 41BB costimulatory domains two days after stimulation with γ-irradiated K562-CD19 cells. Numbers denote the gMFI of PD-1. (b) Unstimulated and (c) stimulated CAR T cells were incubated with Nalm-6-GL-PD-L1 cells at a 1:1,0.3:1,0.1:1 E:T ratio and analyzed using the IncuCyte S3 system. Stimulated CAR T cells were generated by coincubation with Nalm-6-PD-L1-CD80 cells for 6 days prior to cytotoxicity assay. Data are the representative mean ± SD from two independent experiments performed in triplicates. (d) CAR T cells were incubated with γ-irradiated Nalm-6-GL-PD-L1-CD80 or K562-CD19-PD-L1 cells at 1:3 effector: target (E: T) ratio and counted on day 6 after each stimulation. Data are the mean ± SD from two independent experiments performed in triplicates. (e) NSG mice were injected intravenously with 1×10 6 Nalm-6-GL-PD-L1 leukemia cells. 5 days later, 1×10 6 CAR T cells were injected intravenously. Tumor burden was monitored based on the bioluminescence intensity from the IVIS imaging system. Data are from n = 3 mock, n = 5 19G28z and 19GBBz, and n = 4 19P28z and 19PBBz. (f) The number of CAR T cells in mouse blood was determined on day 20 and 43 after CAR T cell injection. Data are mean ± SD from three mice per group. Statistical analysis for (a-d and f) was done by One-Way ANOVA. * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001, ns = not significant.

Article Snippet: Nalm-6-GL, or K562-CD19 cells were transduced to express human PD-L1 (Sino Biological; HG10084-UT cDNA subcloned into a lentiviral vector) to generate Nalm-6-GL-PD-L1 and K562-CD19-PD-L1 cells.

Techniques: Expressing, Irradiation, Incubation, Generated, Cytotoxicity Assay, Injection, Imaging

Journal: bioRxiv

Article Title: Simultaneous, cell-intrinsic downregulation of PD-1 and TIGIT enhances the effector function of CD19-targeting CAR T cells and promotes an early-memory phenotype

doi: 10.1101/2020.11.07.372334

Figure Lengend Snippet: (a) Schematic representation of the generation of CD19-specific CAR T cells with selected shRNA sequences targeting TIM-3, TIGIT, LAG-3, and CTLA-4. ΔLNGFR + T cells were stimulated with γ-irradiated K562-CD19 cells on day 10 and knockdown efficiency was measured on day 12. (b) The expression level of inhibitory receptors in CAR T cells expressing the indicated shRNA cassetttes was evaluated by flow cytometry on day 12. (c) CAR expression levels in CAR T cells expressing the indicated shRNA cassettes were determined on day 10 by flow cytometry. Data are the pooled mean ± SD from two indepdendent experiments performed in duplicates. Statistical analysis was done by One-Way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Nalm-6-GL, or K562-CD19 cells were transduced to express human PD-L1 (Sino Biological; HG10084-UT cDNA subcloned into a lentiviral vector) to generate Nalm-6-GL-PD-L1 and K562-CD19-PD-L1 cells.

Techniques: shRNA, Irradiation, Expressing, Flow Cytometry

Journal: bioRxiv

Article Title: Simultaneous, cell-intrinsic downregulation of PD-1 and TIGIT enhances the effector function of CD19-targeting CAR T cells and promotes an early-memory phenotype

doi: 10.1101/2020.11.07.372334

Figure Lengend Snippet: (a) Schematic representation of the engineered two-in-one vector system carrying dual shRNA cassettes for two ICRs. (b) Dual downregulation efficiency of each ICR in CAR T cells stimulated for 48 hours with γ-irradiated K562-CD19 cells. FACS plots are representative data from two independent experiments performed in duplicates and the bar graphs are the pooled mean ± SD. (c) NSG mice were injected intravenously with 1×10 6 Nalm-6-GL-PD-L1 leukemia cells. 5 days later, 1×10 6 CAR T cells with each dual downregulation (shGFP, shPD-1/shGFP, shPD-1/shTIM-3, shPD-1/shLAG-3, shPD-1/shTIGIT, shPD-1/shCTLA-4) were injected intravenously. Tumor burden was monitored based on the bioluminescence intensity from the IVIS imaging system. Data are from n = 3 mock, shPD-1/shTIM-3 and shPD-1/shLAG-3, n = 4 shGFP, shPD-1/shGFP, and shPD-1/shCTLA-4, n = 5 shPD-1/shTIGIT. (d) Kaplan-Meier survival analysis with the Log-rank (Mantel-Cox) test comparing each CAR T treated mice from (c). (e) NSG mice were injected intravenously with 1×10 6 Nalm-6-GL-PD-L1 leukemia cells. 5 days later, 0.5×10 6 or 0.25×10 6 CAR T cells with PD-1 (shPD-1/shGFP) or PD-1/TIGIT (shPD-1/shTIGIT) downregulation were injected intravenously. Tumor burden was monitored based on the bioluminescence intensity from the IVIS imaging system. Data are from n = 7 mice for the 0.5×10 6 dose groups and n = 6 mice for the 0.25×10 6 dose groups. (f) Kaplan-Meier survival analysis with Log-rank (Mantel-Cox) test comparing CAR T treated mice from (e). Statistical analysis for (b) was done by One-Way ANOVA. * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001 ns, not significant.

Article Snippet: Nalm-6-GL, or K562-CD19 cells were transduced to express human PD-L1 (Sino Biological; HG10084-UT cDNA subcloned into a lentiviral vector) to generate Nalm-6-GL-PD-L1 and K562-CD19-PD-L1 cells.

Techniques: Plasmid Preparation, shRNA, Irradiation, Injection, Imaging

Journal: bioRxiv

Article Title: Simultaneous, cell-intrinsic downregulation of PD-1 and TIGIT enhances the effector function of CD19-targeting CAR T cells and promotes an early-memory phenotype

doi: 10.1101/2020.11.07.372334

Figure Lengend Snippet: (a) The transduction efficiency of dual shRNA constructs was determined by measuring CD19 CAR ΔLNGFR expression by FACS on day 4 after transduction. (b) The gMFI of CD19-CAR in ΔLNGFR + T cells on day 10 after transduction. Data are from the mean ± SD from three donors. (c) CD112 or CD155 expression level in Raji, Nalm-6-GL-PD-L1, K562-CD19-PD-L1, and IM-9 cells with or without IFN-γ treatment for 24 h. CD112 or CD115 expression (d) HLA-DR expression level in Nalm-6-GL-PD-L1 cells with or without IFN-γ treatment at the indicated doses for 24 hours. (e) The intracellular expression level of galectin-9 in Nalm-6-GL-PD-L1 cells. Statistical analysis was done by One-Way ANOVA. ns, not significant.

Article Snippet: Nalm-6-GL, or K562-CD19 cells were transduced to express human PD-L1 (Sino Biological; HG10084-UT cDNA subcloned into a lentiviral vector) to generate Nalm-6-GL-PD-L1 and K562-CD19-PD-L1 cells.

Techniques: Transduction, shRNA, Construct, Expressing

Journal: bioRxiv

Article Title: Simultaneous, cell-intrinsic downregulation of PD-1 and TIGIT enhances the effector function of CD19-targeting CAR T cells and promotes an early-memory phenotype

doi: 10.1101/2020.11.07.372334

Figure Lengend Snippet: (a) Schematic illustration of the vector constructs used to generate 19GBBz, 19PBBz, 19TBBz, and 19PTBBz CAR T cells. (b) Surface expression level of CD19 CAR was evaluated by flow cytometry on day 6 after isolation of transduced cells. Data are the pooled mean ± SD from two independent experiments performed in duplicates. (c) PD-1 and (d) TIGIT expression levels of 19GBBz, 19PBBz, 19TBBz, and 19PTBBz cells stimulated with γ-irradiated K562-CD19 cells for 2 days. Data are the pooled mean ± SD from two indepdendent experiments performed in duplicates. Statistical analysis was done by One-Way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001, ns = not significant.

Article Snippet: Nalm-6-GL, or K562-CD19 cells were transduced to express human PD-L1 (Sino Biological; HG10084-UT cDNA subcloned into a lentiviral vector) to generate Nalm-6-GL-PD-L1 and K562-CD19-PD-L1 cells.

Techniques: Plasmid Preparation, Construct, Expressing, Flow Cytometry, Isolation, Irradiation

Journal: bioRxiv

Article Title: Simultaneous, cell-intrinsic downregulation of PD-1 and TIGIT enhances the effector function of CD19-targeting CAR T cells and promotes an early-memory phenotype

doi: 10.1101/2020.11.07.372334

Figure Lengend Snippet: (a) Schematic representation of CD226 knockout by CRISPR/Cas9 and the flow cytometric evaluation of the expression level of CD226 following knockout by 4 different sgRNA candidates. sgRNA targeting CAG (CMV-IE, chicken actin, rabbit beta globin) was used as a negative control. (b) The knockout efficiency of gRNA #4 targeting CD226 was estimated by T7 endonuclease I assay. (c) Schematic representation of the generation of CD226 KO CD19 specific CAR T cells and the measurement of intracellular IL-2 by flow cytometry, (c) Intracellular IL-2 expression levels in CD226 + and CD226 - populations measured by flow cytometry. Data are the mean ± SD from one experiment performed in triplicates.

Article Snippet: Nalm-6-GL, or K562-CD19 cells were transduced to express human PD-L1 (Sino Biological; HG10084-UT cDNA subcloned into a lentiviral vector) to generate Nalm-6-GL-PD-L1 and K562-CD19-PD-L1 cells.

Techniques: Knock-Out, CRISPR, Expressing, Negative Control, T7EI Assay, Flow Cytometry

Journal: EXCLI Journal

Article Title: Up-regulation of miR-155 potentiates CD34+ CML stem/progenitor cells to escape from the growth-inhibitory effects of TGF-ß1 and BMP signaling

doi: 10.17179/excli2021-3404

Figure Lengend Snippet: A regulatory correlation between miR-155 and TGF-β signaling pathway in CML. A. Mean expression of miR-155 in CD34 + cells from CML patients and normal BM. miRNA level was measured by qRT-PCR and normalized to U6 snRNA as the internal control. B. Transcript levels for SMAD5 and TGF-βR2 in CD34 + cells from CML patients and normal BM. Transcript levels were measured by qRT-PCR and normalized to GAPDH as a housekeeping gene. C, D. SMAD5 and TGF-βR2 are two potential direct targets of miR-155. C. Putative miR-155 binding sites in the 3'-UTR of SMAD5 and TGF-βR2. D. Relative luciferase reporter activity of K562 cells at 48 hr after co-transfection with SMAD5- or TGF-βR2-3'-UTR luciferase reporter constructs and miR-155 precursor molecule (miR-155 mimics). E. miRNA expression level evaluated by qRT-PCR in CD34 + CML cells after 48 hr transfection with miR-155 mimics or negative control scramble (Ctrl). F, G. SMAD5 and TGF-βR2 mRNA expression levels evaluated by qRT-PCR in CD34 + CML cells at 48 hr after transfection with either miR-155 mimics or negative control scramble (Ctrl). SMAD5 and TGF-βR2 mRNA expression levels in CD34 + CML cells 48 hr after transfection with either siRNA against SMAD5 (siSMAD5) or silencing negative control (siCtrl) is also shown. H, I. SMAD5 and TGF-βR2 mRNA expression levels evaluated by qRT-PCR in CD34 + CML cells at 48 hr after transfection with either anti-miR-155 or negative control scramble. Columns, mean of three different experiments; bars, SD. *** P < 0.001.

Article Snippet: Human myeloid cell line K562 was purchased from Pasteur Institute, Tehran, Iran.

Techniques: Expressing, Quantitative RT-PCR, Control, Binding Assay, Luciferase, Activity Assay, Cotransfection, Construct, Transfection, Negative Control

Journal: EXCLI Journal

Article Title: Up-regulation of miR-155 potentiates CD34+ CML stem/progenitor cells to escape from the growth-inhibitory effects of TGF-ß1 and BMP signaling

doi: 10.17179/excli2021-3404

Figure Lengend Snippet: TGF-β1 and BMP signaling activate SMAD5 in CML cells. A. Fold change of SMAD5 in K562 CML cells exposed BMPs or TGF-β1 evaluated by qRT-PCR. Transcript levels were measured by qRT-PCR and normalized to GAPDH as a housekeeping gene. Columns, mean of three different experiments; bars, SD. *** P < 0.001. B. Western blot analysis of phospho-SMAD1/5 levels in CML cells treated with TGF-β1 or BMP2/4, with or without pre-treatment with BMP antagonist dorsomorphin. The TGF-β1 and BMP treatment induced phosphorylation of SMAD 1/5, which are significantly abolished by dorsomorphin. C. Western blot analysis of phospho-SMAD1/5 levels in CML cells treated with the blocking TGF-βR2 antibody. Results showed that TGF-β1-mediated phosphorylation of SMAD1/5 was dramatically abolished following the blocking TGF-βR2 antibody pre-treatment. All Western blot images were representative of at least three independent experiments. The density of bands was assessed using ImageJ software and represented as a relative intensity. Values are means ± SD of three independent experiments. *** P < 0.001.

Article Snippet: Human myeloid cell line K562 was purchased from Pasteur Institute, Tehran, Iran.

Techniques: Quantitative RT-PCR, Western Blot, Phospho-proteomics, Blocking Assay, Software

Journal: Nature communications

Article Title: Simultaneous profiling of chromatin-associated RNA at targeted DNA loci and RNA-RNA Interactions through TaDRIM-seq.

doi: 10.1038/s41467-024-53534-5

Figure Lengend Snippet: Fig. 2 | TaDRIM-seq provides protein-centric RNA-chromatin interactomes, RNA-RNA spatial interactions, and protein-binding DNA and RNA information. a RNA-DNA contact heatmap of chromosome 3 in diverse resolutions by H3K4me3 TaDRIM-seq in K562 cells. Res, resolution. b Region of chromosome 2 showing the interaction of identified RNAs with DNA elements marked by H3K4me3 in K562 cells. c, d RNA-RNA contact heatmap of chromosome 11 at different resolutions in

Article Snippet: Human K562 cells were fixed by 4% paraformaldehyde (Boster; AR1068) spread on slides.

Techniques: Protein Binding