Journal: ImmunoHorizons

Article Title: Chimeric Antigen Cytotoxic Receptors for In Vivo Engineering of Tumor-Targeting NK Cells

doi: 10.4049/immunohorizons.2300099

Figure Lengend Snippet: Design and expression of NK-CARs in peripheral blood NK cells. Left panels indicate schematic representation of NK-CAR designs with different lengths of their ECD; center panels indicate dot plots for NK-CAR cell surface expression in human peripheral blood-activated NK cells 20 h after electroporation. The graph in the right panels indicate the percent CAR positive for the different NK-CAR constructs for ( A ) NKp30-based CAR, ( B ) NKp44-based CAR, and ( C ) NKp46-based CAR. Representative data of three independent experiments using two independent donors are shown. ECD, extracellular domain; SP, signal peptide.

Article Snippet: A feeder cell–free method using an ImmunoCult NK cell expansion kit (STEMCELL Technologies) was used to activate isolated NK cells in vitro for 6 d. The isolated NK cells were resuspended in ImmunoCult NK expansion media and seeded on plates coated with ImmunoCult NK cell expansion coating material for 6 d at 37°C in a cell culture incubator.

Techniques: Expressing, Electroporation, Construct

Journal: ImmunoHorizons

Article Title: Chimeric Antigen Cytotoxic Receptors for In Vivo Engineering of Tumor-Targeting NK Cells

doi: 10.4049/immunohorizons.2300099

Figure Lengend Snippet: Antitumor activities of NK-CAR constructs. ( A ) Cell surface expression of NK-CARs in human peripheral blood-activated NK cells electroporated with NK-CAR mRNA detected by surface labeling with dye-conjugated recombinant HER2 protein. ( B ) Quantification of flow cytometry plot shown in (A). ( C ) Tumoricidal activity of HER2-targeting NK-CARs. NK cells were cocultured with HER2 + SKOV3 tumor cells at a 5:1 E:T ratio and incubated for 20 h at 37°C. The graph represents the percentage specific killing of SKOV3 cells by NK-CAR–transfected NK cells compared with mock-transfected NK cells. Data are representative of three independent experiments with two donors. The mean ± SD is plotted on the graph, and statistical significance was determined by an ordinary one-way ANOVA with a Dunnett multiple comparison test between mock-transfected NK cells versus NK-CAR cells. The p values are indicated on the graph. ( D ) NK-CARs induce cytokine (TNF-α, IFN-γ, and GM-CSF) and chemokine (CCL2) production when activated by coculturing with SKOV3 tumor cells. The mean ± SD from three replicates is shown. Statistical significance was determined by two-way ANOVA with a Dunnett multiple comparison test between mock and SKOV3 coculture versus NK-CAR and SKOV3 coculture. The p values are indicated on the graph. Data are representative of two independent experiments.

Article Snippet: A feeder cell–free method using an ImmunoCult NK cell expansion kit (STEMCELL Technologies) was used to activate isolated NK cells in vitro for 6 d. The isolated NK cells were resuspended in ImmunoCult NK expansion media and seeded on plates coated with ImmunoCult NK cell expansion coating material for 6 d at 37°C in a cell culture incubator.

Techniques: Construct, Expressing, Labeling, Recombinant, Flow Cytometry, Activity Assay, Incubation, Transfection, Comparison