Journal: Frontiers in Immunology

Article Title: Bryostatin Activates CAR T-Cell Antigen-Non-Specific Killing (CTAK), and CAR-T NK-Like Killing for Pre-B ALL, While Blocking Cytolysis of a Burkitt Lymphoma Cell Line

doi: 10.3389/fimmu.2022.825364

Figure Lengend Snippet: Viability and Surface Expression of CD19 and CD22 in treated B-cell leukemia cell lines and normal B cells. Left panel: Epigenetic modifiers/differentiation agents (Bryostatin, 5-Azacytidine, ATRA, Panobinostat or Vorinostat) were added at increasing concentrations (x-axis, as indicated) to the culture media of B cell lines (Raji-green circle, NALM6-magenta square, REH-blue triangle) for 48 hours (or 24 h for bryostatin). Following drug exposure, cell viability was calculated and plotted (column 1). Each agent adversely affected viability as concentration increased, except for bryostatin. Surface expression of CD19 (column 2) and CD22 (column 3) in leukemia cell lines was qualified by flow cytometry using Quanti-Brite PE beads. Average of triplicate wells is shown, values differing from untreated controls are indicated, * indicates p<0.05. Right panel: Expanded peripheral blood B cells from three donors, cultured on CD40L expressing feeder cells in media supplemented with (squares) or without (circles) B cell growth factors (IL-2, IL-4, IL-21, BAFF, see Materials and Methods ), were tested for changes in cell surface expression induced by bryostatin. The number of CD19 and CD22 molecules differed between donors to a degree, but was not significantly impacted by bryostatin, paired t-test p>0.05, grand median, solid bar, shown for reference.

Article Snippet: T cells were phenotyped with: anti-CD3 (BioLegend, clone HIT3a, PB), CD4 (BioLegend, clone SK3, FITC), CD8 (BD Biosciences, clone RPAT8, BUV395), biotinylated CD22 protein (Sino Biological, for CAR detection) and SA-PE (BioLegend).

Techniques: Expressing, Concentration Assay, Flow Cytometry, Cell Culture

Journal: Frontiers in Immunology

Article Title: Bryostatin Activates CAR T-Cell Antigen-Non-Specific Killing (CTAK), and CAR-T NK-Like Killing for Pre-B ALL, While Blocking Cytolysis of a Burkitt Lymphoma Cell Line

doi: 10.3389/fimmu.2022.825364

Figure Lengend Snippet: Total RNA and protein levels of CD19 and CD22 in bryostatin treated leukemia cell lines. Western blot analysis of (A) CD19 and (B) CD22 protein expression in Raji, NALM6 and REH cell lines with (+B) or without bryostatin treatment. (C) CD19 and (D) CD22 band intensity from three independent experiments was quantified and normalized to β-actin. For each line, treated and non-treated groups were compared. There was no significant difference (ns) between groups of at the protein level. RNA levels for (E) CD19 and (F) CD22 were quantified by ddRT-PCR. CD19 and CD22 copies per ng RNA were calculated and analyzed. Significant differences between treated and untreated groups were seen for CD22 in Raji cells ( p < 0.05 ). *p < 0.05. ns, not significant.

Article Snippet: T cells were phenotyped with: anti-CD3 (BioLegend, clone HIT3a, PB), CD4 (BioLegend, clone SK3, FITC), CD8 (BD Biosciences, clone RPAT8, BUV395), biotinylated CD22 protein (Sino Biological, for CAR detection) and SA-PE (BioLegend).

Techniques: Western Blot, Expressing

Journal: Frontiers in Immunology

Article Title: Bryostatin Activates CAR T-Cell Antigen-Non-Specific Killing (CTAK), and CAR-T NK-Like Killing for Pre-B ALL, While Blocking Cytolysis of a Burkitt Lymphoma Cell Line

doi: 10.3389/fimmu.2022.825364

Figure Lengend Snippet: Surface expression of CD19 and CD22 upon co-culture with anti-CD22 CAR-T. Using Quanti-Brite analysis, the number of CD19 and CD22 molecules (y-axis) on the surface of Raji, NALM6, and REH cell lines was quantified, following co-culture with CD22 CAR-T, at the indicated effector to target ratios, x-axis. The leftmost pair of columns quantifies surface expression on untreated cell lines. Significant differences from control are shown *p < 0.05. The x-axis lists the cell line tested, exposure to bryostatin (B, magenta bars) or CD22 CAR-T alone (green).

Article Snippet: T cells were phenotyped with: anti-CD3 (BioLegend, clone HIT3a, PB), CD4 (BioLegend, clone SK3, FITC), CD8 (BD Biosciences, clone RPAT8, BUV395), biotinylated CD22 protein (Sino Biological, for CAR detection) and SA-PE (BioLegend).

Techniques: Expressing, Co-Culture Assay

Journal: Frontiers in Immunology

Article Title: Bryostatin Activates CAR T-Cell Antigen-Non-Specific Killing (CTAK), and CAR-T NK-Like Killing for Pre-B ALL, While Blocking Cytolysis of a Burkitt Lymphoma Cell Line

doi: 10.3389/fimmu.2022.825364

Figure Lengend Snippet: Surface expression of CD19 and CD22 following bryostatin wash-out and CAR-T removal. After overnight culture with anti-CD22 CAR-T, the number of cell surface proteins was quantified using Quanti-Brite analysis, average of triplicate wells and standard deviations are shown. 0 hr, x-axis, is after the overnight culture, and each time point represents cell surface proteins on the surface of untreated Raji, NALM6, or REH (Leukemia cell, green bars), treated with bryostatin alone (Leukemia + Bryostatin, magenta bars), treated with CAR-T alone (Leukemia+CART, blue bars), or treated with both CAR-T and bryostatin (Leukemia+CART+Bryostatin, red bars), at the time points listed, x-axis. Significant differences from leukemia alone are shown *p < 0.05.

Article Snippet: T cells were phenotyped with: anti-CD3 (BioLegend, clone HIT3a, PB), CD4 (BioLegend, clone SK3, FITC), CD8 (BD Biosciences, clone RPAT8, BUV395), biotinylated CD22 protein (Sino Biological, for CAR detection) and SA-PE (BioLegend).

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: Bryostatin Activates CAR T-Cell Antigen-Non-Specific Killing (CTAK), and CAR-T NK-Like Killing for Pre-B ALL, While Blocking Cytolysis of a Burkitt Lymphoma Cell Line

doi: 10.3389/fimmu.2022.825364

Figure Lengend Snippet: Transfer of CD19 and CD22 to CD22 CAR-T following overnight culture with leukemia cell lines. The number of CD19 and CD22 molecules acquired by anti-CD22 CAR-T (trogocytosis) was quantified using Quanti-Brite analysis, as per Figure 3 , however in this case the T cells were analyzed. Average antigen expression and standard deviation are shown. Background signal is shown as a gray bar for each subgroup (CART). The x-axis lists the anti-CD22 CAR-T to leukemia cell ratio (E:T) used for each condition and indicates if the leukemia line had been treated with bryostatin (B, magenta bars).

Article Snippet: T cells were phenotyped with: anti-CD3 (BioLegend, clone HIT3a, PB), CD4 (BioLegend, clone SK3, FITC), CD8 (BD Biosciences, clone RPAT8, BUV395), biotinylated CD22 protein (Sino Biological, for CAR detection) and SA-PE (BioLegend).

Techniques: Expressing, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Bryostatin Activates CAR T-Cell Antigen-Non-Specific Killing (CTAK), and CAR-T NK-Like Killing for Pre-B ALL, While Blocking Cytolysis of a Burkitt Lymphoma Cell Line

doi: 10.3389/fimmu.2022.825364

Figure Lengend Snippet: Anti-CD22 CAR-T mediated cellular cytotoxicity (CTL) of bryostatin-treated leukemia. (A–D) Average lysis from triplicate wells for four cell lines (Raji, NALM6, REH, and K562) by anti-CD22 CART (CD22 CART, open circle) or un-transduced T cells from the same donor (UTD, open triangle), treated with bryostatin (+B, closed shape) or untreated (open shape), a the E:T ratios listed on the x-axis. (E–H) Assay tested in parallel including cold-target inhibition (addition of K562 at a 30:1 E:T ratio). Representative results for T cells from 3 donors are shown, each data point showing the average and standard deviation from three replicate wells.

Article Snippet: T cells were phenotyped with: anti-CD3 (BioLegend, clone HIT3a, PB), CD4 (BioLegend, clone SK3, FITC), CD8 (BD Biosciences, clone RPAT8, BUV395), biotinylated CD22 protein (Sino Biological, for CAR detection) and SA-PE (BioLegend).

Techniques: Lysis, Inhibition, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Bryostatin Activates CAR T-Cell Antigen-Non-Specific Killing (CTAK), and CAR-T NK-Like Killing for Pre-B ALL, While Blocking Cytolysis of a Burkitt Lymphoma Cell Line

doi: 10.3389/fimmu.2022.825364

Figure Lengend Snippet: Bryostatin treatment reveals multiple pathways that CAR-T cells use to eliminate leukemia. In the center of the diagram, pre-B ALL cells are illustrated, displaying the CAR-T target antigen, CD22, innate immune receptor ligands induced by bryostatin that are recognized by activated T cells (Bryostatin-induced NK ligands) and ligands recognized by T cells that have been: a) sensitized by CAR-T production, b) bryostatin-induced, and c) not blocked by cold-target inhibition (CTAK, CAR-T cell non-antigen-specific killing). Also shown are a non-overlapping set of alternative innate immune receptor ligands that are recognized by NK92 upon bryostatin-treatment (right-most effector cell). Cold-target inhibition does not affect NK92 or CD22-specific CAR-T killing. Cold-target does decrease killing evidenced by activated T cells (UTD), but incompletely for CTAK-mediated killing. Green arrows indicate successful cytolysis and blunt red arrow indicates killing impacted by K562-mediated cold target inhibition (classic NK killing).

Article Snippet: T cells were phenotyped with: anti-CD3 (BioLegend, clone HIT3a, PB), CD4 (BioLegend, clone SK3, FITC), CD8 (BD Biosciences, clone RPAT8, BUV395), biotinylated CD22 protein (Sino Biological, for CAR detection) and SA-PE (BioLegend).

Techniques: Inhibition

Journal: Frontiers in Immunology

Article Title: Bryostatin Activates CAR T-Cell Antigen-Non-Specific Killing (CTAK), and CAR-T NK-Like Killing for Pre-B ALL, While Blocking Cytolysis of a Burkitt Lymphoma Cell Line

doi: 10.3389/fimmu.2022.825364

Figure Lengend Snippet: Impact of EBV lytic cycle inhibition on CD22 CAR-T mediated killing of bryostatin-treated Raji leukemia cells. Raji cells were cultured for 2 weeks in the presence or absence of 15 uM ganciclovir (+Ganciclovir in legend), and for the final day of culture bryostatin was added where indicated (+Bryo in legend). Treated cells were then used as targets in CTL assays using anti-CD22 CAR-T (CART) or untransduced T cells (UTD) as effector cells. Average cytolysis of 3 replicate wells is plotted for each condition. Results are representative of three independent experiments.

Article Snippet: T cells were phenotyped with: anti-CD3 (BioLegend, clone HIT3a, PB), CD4 (BioLegend, clone SK3, FITC), CD8 (BD Biosciences, clone RPAT8, BUV395), biotinylated CD22 protein (Sino Biological, for CAR detection) and SA-PE (BioLegend).

Techniques: Inhibition, Cell Culture

Journal: Frontiers in Immunology

Article Title: Bryostatin Activates CAR T-Cell Antigen-Non-Specific Killing (CTAK), and CAR-T NK-Like Killing for Pre-B ALL, While Blocking Cytolysis of a Burkitt Lymphoma Cell Line

doi: 10.3389/fimmu.2022.825364

Figure Lengend Snippet: Blocking of innate immunoreceptor ligands during CAR-T mediated cytolysis. The NALM6 pre-B ALL cell line was exposed to anti-CD22 CAR-T or control UTD cells at an E:T ratio of 10:1, y-axis. Results are grouped in each panel by cytolysis seen with untreated target (gray, control), bryostatin treatment (black), or treated with blocking agent (pink) or blocking agent and bryostatin (purple), using (A) recombinant DNAM-1, (B) NKG2D, (C) NKp30, or (D) anti-ICAM1 antibody, for 30 minutes prior to addition of effector cells. Average of 3 replicate wells are shown, with statistical difference between groups plotted, ns p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001. (E–H) REH leukemia cells were similarly analyzed.

Article Snippet: T cells were phenotyped with: anti-CD3 (BioLegend, clone HIT3a, PB), CD4 (BioLegend, clone SK3, FITC), CD8 (BD Biosciences, clone RPAT8, BUV395), biotinylated CD22 protein (Sino Biological, for CAR detection) and SA-PE (BioLegend).

Techniques: Blocking Assay, Recombinant

Journal: Journal for Immunotherapy of Cancer

Article Title: T cell-specific non-viral DNA delivery and in vivo CAR-T generation using targeted lipid nanoparticles

doi: 10.1136/jitc-2025-011759

Figure Lengend Snippet: NCtx-dual induces robust and durable in vivo CAR-T generation, tumor control and extended survival in CD34+ HSC-engrafted NCG mice. ( a ) Schematic representation of study design: NCG mice engrafted with CD34+ HSC (NCG-His) were injected intravenously with 5×10 5 luciferase-expressing Nalm6 tumor cells, followed by IP injection of 200 ng IL-7. Mice were treated intravenously with NCtx-dual or a NCtx vehicle control encapsulating eGFP mcDNA and SB100x mRNA (vehicle control) at a total nucleic acid dose of 50 µg/kg. ( b ) CD19/CD22 dual CAR mcDNA expression was assessed by flow cytometry in circulating T cells for 40 days post-NCtx administration. n=12, data are presented as mean with individual values. ( c ) Nalm6 tumor burden was monitored by BLI. ( d ) Kaplan-Meier survival analysis. n=6 (vehicle control) or n=12 (NCtx-dual). ( e ) Expression of the exhaustion marker PD-1 in CAR+ and CAR− T cell populations over time in NCtx-dual-treated mice, analyzed by flow cytometry. n=12, data represent mean±individual values. ( f ) T cell phenotype characterization (Tnaive/Tscm, Tcm, Tem, and Teff) based on CD45RA and CD62L expression in CAR+ and CAR− T cells after NCtx-dual administration. n=12, data represent mean±SD. P values were calculated using log-rank Mantel-Cox test ( b ) or two-way ANOVA, mixed effect model ( d, e ). Significance is plotted with ns for p>0.0332 and *p<0.0332. ANOVA, analysis of variance; BLI, bioluminescent imaging; CAR, chimeric antigen receptor; HSC, hematopoietic stem cell; IL-7, interleukin 7; IP, intraperitoneal; mcDNA, minicircle DNA; mRNA, messenger RNA; ns, not significant; PD-1, programmed cell death protein-1; Tcm, central memory T cell; Teff, effector T cell; Tem, effector memory T cell; Tnaive, naïve T cell; Tscm, stem cell memory T cell.

Article Snippet: CD19/CD22 dual CAR expression was detected using CD22 CAR detection reagent (Miltenyi Biotec, REA130-126-727, 1:1000) and streptavidin-APC-Cy7 (BD, REA746, 1:200).

Techniques: In Vivo, Control, Injection, Luciferase, Expressing, Flow Cytometry, Marker, Imaging

Journal: Cancers

Article Title: Engineering Induced Pluripotent Stem Cells for Cancer Immunotherapy

doi: 10.3390/cancers14092266

Figure Lengend Snippet: Current allogeneic cell therapies in clinical trials.

Article Snippet: NCAT041500497 , Phase 1 study of UCART22 in patients with R/R CD22+ BALL , Allogeneic T cells expressing anti-CD22 CAR , Relapsed or refractory CD22 + B-cell acute lymphoblastic leukemia , Cellectis.

Techniques: Bioprocessing, Expressing, Activity Assay