addgene plasmid  (Addgene inc)

Journal: bioRxiv

Article Title: Site-specific genome engineering of primary human natural killer cells for programmable anti-tumor function

doi: 10.1101/2025.10.05.680386

Figure Lengend Snippet: Three multicolor flow cytometry panels were designed using bright fluorophores and compatible with the fluorescent proteins expressed by tumor cell lines (GFP, mKate2), allowing to investigate the expression of the main NK receptor ligands on their surface. a , To compare different cell lines while accounting for differences in background noise, a staining index (SI) was used for each ligand. The SI definition was based on the Mean Fluorescence Intensity (MFI) of the sample and the respective FMO used as negative control, and on the standard deviation σ of the FMO. b , Example of the staining obtained for CD112 for different cell lines and the corresponding Staining Index. c , Heatmap representing the expression (Staining Index) for different NK ligands. Each value represents the mean of 2 technical replicates. The main NK receptors known to bind the corresponding tumor ligands are indicated on the left. The + indicates when there is saturation of the staining index based on the color scale used for the heatmap.

Article Snippet: Clonally selected firefly luciferase + mKate2 + NGFR + K562 cells and firefly luciferase + blasticidin resistant RAJI cells (ATCC) were cultured in RPMI-1640 (2 mM L-glutamine) supplemented with FBS (10%) and penicillin-streptomycin (1%).

Techniques: Flow Cytometry, Expressing, Staining, Fluorescence, Negative Control, Standard Deviation

Journal: Blood Cancer Discovery

Article Title: DLBCL Cells Emerge after CD19 CAR T Cells with Cross-Antigen Resistance and a Gene Signature Predictive of Clinical CAR T-cell Response

doi: 10.1158/2643-3230.BCD-24-0176

Figure Lengend Snippet: Cancer cells are differentially responsive to CAR T-cell killing. A, CD19 CAR T-cell sensitivity of DLBCL, ALL, and CLL cell lines ( n = 15, n = 2, n = 2) ranked from most to least sensitive. Cancer cells lines expressing the fluorescent protein mKate2 were cocultured with CD19 CAR T cells at a 0.375:1, 1.5:1, or 6:1 E:T ratio. Heatmap represents growth of cancer cells over a time course of 4 days measured by fluorescent imaging of mKate2 signal normalized to timepoint zero. Mean of n = 4 technical replicates are shown. Red = growth and blue = death. B, MFI of CD19 cell surface antigen expression measured by flow cytometry in DLBCL, ALL, and CLL cell lines ranked from the most to least CD19 CAR T cell–sensitive. Mean + SD of n = 3 technical replicates are shown. C, Correlation between CD19 cell surface expression (MFI) and CD19 CAR T-cell sensitivity (AUC of 1.5:1 E:T ratio normalized to AUC of UTD treatment). Data points represent n = 19 cell lines. D, No correlation between CD20 cell surface expression (MFI) and CD20 CAR T-cell sensitivity (AUC of 1.5:1 E:T ratio normalized to AUC of UTD treatment). Data points represent n = 19 cell lines. E, Strong correlation of response to CD19 and CD20 CAR T-cell treatment. Data points represent n = 14 cell lines. CD19- and CD20-negative cell lines (flow cytometry MFI < 100) were excluded from the analysis. CLL, chronic lymphocytic B-cell leukemia.

Article Snippet: To assess CAR T-cell sensitivity as well as generate CAR T cell–resistant cell lines, mKate2 + cancer cells (stably transduced with Incucyte Nuclight Red Lentivirus, Sartorius, 4476) or GFP + cancer cells (stably transduced with Incucyte Nuclight Green Lentivirus, Sartorius, 4475) were cultured with CAR T cells at multiple E:T ratios in a 1:1 mixture of cancer cell medium and T-cell medium (RPMI based as described above) in a 96-well (all long-term cocultures) or 384-well plate format.

Techniques: Expressing, Imaging, Flow Cytometry

Journal: Blood Cancer Discovery

Article Title: DLBCL Cells Emerge after CD19 CAR T Cells with Cross-Antigen Resistance and a Gene Signature Predictive of Clinical CAR T-cell Response

doi: 10.1158/2643-3230.BCD-24-0176

Figure Lengend Snippet: BH3-mimetics sensitize DLBCL cells to CAR T-cell killing. A, Small-molecule screen identified Mcl-1 inhibitor and Bcl-2 inhibitor as top CAR T-cell sensitizers. Five mKate2 + DLBCL cell lines were cocultured with CAR19-GFP CAR T cells at a 1.2:1 (RL and WSU-DLCL2) or 0.4:1 (DoHH2, SU-DHL-6, and TMD8) E:T ratio in presence of small-molecule compounds for 4 days. Heatmap represents the average growth of cancer cells treated with CAR T cells and small-molecule compounds at concentrations of 1.111 and 0.370 μmol/L normalized to the growth of cancer cells treated with CAR T cells alone. Dark blue: reduced cancer cell numbers/synergistic combination. Red: increased cancer cell growth. Median of n = 3 + 3 technical triplicates are shown. B, BH3 mimetics sensitize DLBCL cell lines to CAR T-cell killing. Selected DLBCL cell lines were cocultured with CD19 CAR T cells (1:1 E:T ratio) and 300 nmol/L Mcl-1 inhibitor tapotoclax or 300 nmol/L Bcl-2 inhibitor venetoclax for 3 days. Heatmap represents cell growth at day three normalized to timepoint zero. Mean of n = 4 technical replicates are shown. Max = maximum value specific to each cell line. C, MCL1 and BCL2 mRNA expression levels in UPCC-13413 DLBCL patient pretreatment biopsies showing a trend toward higher MCL1 expression nonresponders (patients with PD, n = 14) vs. complete responders (CR, n = 7). D, mRNA expression levels of the effector caspases CASP3 , CASP6 , and CASP7 and the initiator caspase CASP9 in pretreatment biopsies from UPCC-13413 patients with DLBCL show significantly lower expression of CASP6 and slightly higher expression of CASP9 in nonresponders (PD, n = 14) vs. complete responders (CR, n = 7). *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; two-tailed unpaired t test. CPM, counts per million.

Article Snippet: To assess CAR T-cell sensitivity as well as generate CAR T cell–resistant cell lines, mKate2 + cancer cells (stably transduced with Incucyte Nuclight Red Lentivirus, Sartorius, 4476) or GFP + cancer cells (stably transduced with Incucyte Nuclight Green Lentivirus, Sartorius, 4475) were cultured with CAR T cells at multiple E:T ratios in a 1:1 mixture of cancer cell medium and T-cell medium (RPMI based as described above) in a 96-well (all long-term cocultures) or 384-well plate format.

Techniques: Expressing, Two Tailed Test

Journal: Blood Cancer Discovery

Article Title: DLBCL Cells Emerge after CD19 CAR T Cells with Cross-Antigen Resistance and a Gene Signature Predictive of Clinical CAR T-cell Response

doi: 10.1158/2643-3230.BCD-24-0176

Figure Lengend Snippet: Cancer cells acquire resistance to CAR T-cell killing with clinically relevant mechanisms. A, Schematic illustration of experimental setup. mKate2 + DLBCL cell lines were cocultured with CD19 CAR T cells, and cancer cell growth was monitored by live cell imaging. Cancer cells that grew out from cocultures were expanded and rechallenged with fresh CAR T cells to identify cell lines that have become inherently resistant. B, Outgrowth of SU-DHL-10 (left) and SU-DHL-5 (right) cells from long-term cocultures with CD19 CAR T cells at a 0.4:1 or 1.2:1 E:T ratio. Lines represent growth of cancer cells for n = 6 replicates per condition. C, Rechallenge of SU-DHL-10 (top) and SU-DHL-5 (bottom) cells that grew out from long-term cocultures. Cancer cells were cocultured with CAR T cells at 0.4:1, 1.2:1, and 4:1 E:T ratios. Heatmap represents growth of cancer cells over a time course of 6 days normalized to timepoint zero. Mean of n = 2 technical replicates per sample is shown. Red = growth and blue = death. Samples marked with asterisk were kept in coculture until cells grew out, from which additional resistant cell lines were established. D, Decline of CD19 and CD58 expression in CAR T cell–resistant SU-DHL-10 and SU-DHL-5 cell lines. Representative flow cytometry plots for selected cell lines that had grown out from cocultures once or twice are shown. Cell lines had been exposed to different degrees of selective pressure exerted by a high E:T ratio or multiple exposures to CAR T cells as indicated. Low selective pressure: cells 1× cocultured at a low E:T ratio (light blue). Intermediate selective pressure: cells 2× cocultured with increasing E:T ratios (dark blue) or cells 1× cocultured at a high E:T ratio (light pink). High selective pressure: cells 2× cocultured at a high E:T ratio (dark pink). Normalization mode: unit area.

Article Snippet: To assess CAR T-cell sensitivity as well as generate CAR T cell–resistant cell lines, mKate2 + cancer cells (stably transduced with Incucyte Nuclight Red Lentivirus, Sartorius, 4476) or GFP + cancer cells (stably transduced with Incucyte Nuclight Green Lentivirus, Sartorius, 4475) were cultured with CAR T cells at multiple E:T ratios in a 1:1 mixture of cancer cell medium and T-cell medium (RPMI based as described above) in a 96-well (all long-term cocultures) or 384-well plate format.

Techniques: Live Cell Imaging, Expressing, Flow Cytometry