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: Cancers

Article Title: Folic Acid-Appended Hydroxypropyl-β-Cyclodextrin Exhibits Potent Antitumor Activity in Chronic Myeloid Leukemia Cells via Autophagic Cell Death.

doi: 10.3390/cancers13215413

Figure Lengend Snippet: Figure 3. Antitumor effect of FA-HP-β-CyD. (a,b) Antitumor activity of FA-HP-β-CyD (0, 0.05, 0.1, 0.25, 0.5, and 1.0 mM) in K562 and BV173 cells. Cells were incubated with FA-HP-β-CyD for 72 h at 37 ◦C. (c) Antitumor activity of FA-HP-β-CyD and HP-β-CyD in A549 cells. Cells were incubated with 0, 2.5, 5, 7.5, and 10 mM of FA-HP-β-CyD or HP-β-CyD for 72 h at 37 ◦C. (d,e) Treatment with FA-HP-β-CyD in the presence or absence of FA. K562 and BV173 cells were incubated for 2 h at 37 ◦C with medium only (control), medium containing FA-HP-β-CyD (5 mM) and HP-β-CyD (5 mM) in the absence and presence of FA (2 mM). Each value represents the mean ± SEM of three experiments. * p < 0.05. The cell number ratio is the number of cells calculated with the number of cells in the control as 1.

Article Snippet: The human CML cell lines BV173 and K562 were purchased from DSMZ (Braunschweig, Germany) and the American Type Culture Collection (ATCC, Manassas, VA, USA), respectively.

Techniques: Activity Assay, Incubation, Control

Journal: Cancers

Article Title: Folic Acid-Appended Hydroxypropyl-β-Cyclodextrin Exhibits Potent Antitumor Activity in Chronic Myeloid Leukemia Cells via Autophagic Cell Death.

doi: 10.3390/cancers13215413

Figure Lengend Snippet: Figure 4. FA-HP-β-CyD induces apoptosis in K562 cells, BV173 cells, and Ba/F3BCR-ABL cells. (a) K562 cells, BV173 cells, and Ba/F3BCR-ABL cells were treated with 0, 0.5, 1.0, and 1.5 mM of FA-HP-β-CyD. After 72 h of culture, the Annexin V and PI-staining was done. Representative FACS plots are shown (n = 3). (b–d) Percentages of Annexin V-positive PI-negative cells exposed to FA-HP-β-CyD for 72 h are shown. Data represent the mean ± SD of three independent experiments. * p < 0.05.

Article Snippet: The human CML cell lines BV173 and K562 were purchased from DSMZ (Braunschweig, Germany) and the American Type Culture Collection (ATCC, Manassas, VA, USA), respectively.

Techniques: Staining

Journal: Cancers

Article Title: Folic Acid-Appended Hydroxypropyl-β-Cyclodextrin Exhibits Potent Antitumor Activity in Chronic Myeloid Leukemia Cells via Autophagic Cell Death.

doi: 10.3390/cancers13215413

Figure Lengend Snippet: Figure 5. Intracellular distribution of FA-HP-β-CyD. (a) Cellular association of TRITC-FA-HP-β-Cy in K562 cells (left) and BV173 cells (right). The fluorescence intensity derived from TRITC was determined by flow cytometry at 1 h after incubation at 37 ◦C. Control: medium only. (b,c) Intra- cellular distribution of TRITC-FA-HP-β-CyD. CML cells were treated with medium only (control), TRITC-FA-HP-β-CyD (1 mM), TRITC-FA-HP-β-CyD (1 mM) and FA (2 mM), or TRITC-HP-β-CyD (10 mM) for 1 h. The experiments were performed three times independently and representative images are shown.

Article Snippet: The human CML cell lines BV173 and K562 were purchased from DSMZ (Braunschweig, Germany) and the American Type Culture Collection (ATCC, Manassas, VA, USA), respectively.

Techniques: Derivative Assay, Cytometry, Incubation, Control

Journal: Cancers

Article Title: Folic Acid-Appended Hydroxypropyl-β-Cyclodextrin Exhibits Potent Antitumor Activity in Chronic Myeloid Leukemia Cells via Autophagic Cell Death.

doi: 10.3390/cancers13215413

Figure Lengend Snippet: Figure 6. Induction of autophagy by treatment with FA-M-β-CyD. (a) Detection of autophagy in CML cells. K562 cells treated with FA-HP-β-CyD and HP-β-CyD for 2 h were exposed to Cyto-ID for 30 min. For observation, fluorescence microscopy was used. (b) Effect of HP-β-CyDs on LC3B expression in K562 cells. Cells were treated with medium only (control), FA-HP-β-CyD, HP-β-CyD, imatinib (IM), and rapamycin (Rap) for 2 h. LC3B protein levels were detected by western blotting. (c) The graph shows the fluorescence intensity of the bands. * p < 0.05 compared with the control. (d,e) Effects of chloroquine, bafilomycin A1, and LY294002 on the antitumor activity of FA-HP-β-CyD (d) and HP-β-CyD (e) in BV173 cells. Cells were incubated for 24 h. * p < 0.05 compared with FA-HP-β-CyD.

Article Snippet: The human CML cell lines BV173 and K562 were purchased from DSMZ (Braunschweig, Germany) and the American Type Culture Collection (ATCC, Manassas, VA, USA), respectively.

Techniques: Microscopy, Expressing, Control, Western Blot, Activity Assay, Incubation

Journal: Cancers

Article Title: Folic Acid-Appended Hydroxypropyl-β-Cyclodextrin Exhibits Potent Antitumor Activity in Chronic Myeloid Leukemia Cells via Autophagic Cell Death.

doi: 10.3390/cancers13215413

Figure Lengend Snippet: Figure 7. Detection of mitophagy, measurement of intracellular ATP levels, and ROS generation assay in CML cells. (a,c) Detection of mitophagy in K562 and BV173 cells. The cells were treated with Mtphagy dye, a mitophagy detection reagent, and then incubated with medium only (control), 1 mM of FA-HP-β-CyD, and 10 mM of HP-β-CyD, followed by Lyso dye, a lysosome detection reagent. Representative images are shown (n = 3). (b,d) Fluorescence intensities of Mtphagy dye and Lyso dye in the control, in HP-β-CyD-treated, and in FA-HP-β-CyD-treated cells are shown in bar graphs. * p < 0.05 compared with the control. (e) Measurement of intracellular ATP levels. K562 and BV173 cells were incubated with medium only (control), 1 mM of FA-HP-β-CyD, and 10 mM of HP-β-CyD for 2 h. Then, the cells were treated with ATP detection reagent. Bar graphs represent the mean ± SEM (n = 3 per group). * Significant difference with p < 0.05 compared with the control and FA-HP-β-CyD. (f) ROS generation assay. K562 and BV173 cells were incubated with medium only (control), 1 mM of FA-HP-β-CyD, and 10 mM of HP-β-CyD, and detected by flow cytometry using ROS detection reagents. Blue: control; orange: HP-β-CyD; and red: FA-HP-β-CyD.

Article Snippet: The human CML cell lines BV173 and K562 were purchased from DSMZ (Braunschweig, Germany) and the American Type Culture Collection (ATCC, Manassas, VA, USA), respectively.

Techniques: Incubation, Control, Fluorescence, Cytometry

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

Journal: The Journal of Immunology Author Choice

Article Title: Human Fc?RII Cytoplasmic Domains Differentially Influence Antibody-Mediated Dengue Virus Infection

doi: 10.4049/jimmunol.1203052

Figure Lengend Snippet: Influence of FcγRIIa and FcγRIIb isoforms on ADE of dengue virus infection. (A) Representative phenotype of FcγRIIa expression on the cell surface of K562 cells following treatment with FcγRIIa (CD32a; red) or nonspecific siRNA control (green) compared with nontransfected K562 (blue) from at least three independent experiments. (B) ADE of dengue virus infection on wild type, FcγRIIa siRNA, or control siRNA–treated K562 cells (upper panel) and FcγRIIa specific Ab treated K562 cell (lower panel). (C) Expression of FcγRIIa and FcγRIIb on K562 cells transfected with hFcγRIIa cDNA or pCMV6-XL4 control plasmid. (D) Percent DV infection in FcγRIIb transfected and control transfected K562 cells in the absence or presence of dengue immune serum at enhancement titers. Data are shown as mean ± SE from n = 3 independent experiments.

Article Snippet: Next, we transfected FcγRIIb cDNA (Origene, Rockville, MD) into K562 and observed nearly 40% FcγRIIb surface expression at 24 h posttransfection using flow cytometry ( , lower right panel ).

Techniques: Infection, Expressing, Transfection, Plasmid Preparation