Journal: Cancers

Article Title: Cytolytic Activity of CAR T Cells and Maintenance of Their CD4+ Subset Is Critical for Optimal Antitumor Activity in Preclinical Solid Tumor Models

doi: 10.3390/cancers13174301

Figure Lengend Snippet: EM-enriched but not CM-enriched HER2-CAR T cells eliminate HER2-positive tumor xenografts in vivo. Mice were injected s.c. with 3 × 10 6 JIMT-1.ffLuc cells. Mice on day 14 (arrow) received a single i.v. dose of 2.5 × 10 6 NT T cells (NT OKT3-antiCD28/RPMI T cells, n = 4, grey, straight line; NT OKT3-RetroNectin/LymphoONE T cells, n = 5, grey, dashed line) or HER2-CAR T cells (CD28.z OKT3-antiCD28/RPMI T cells, n = 5, pink, straight line; CD28.z OKT3-RetroNectin/LymphoONE T cells, n = 5, pink, dashed line; 41BB.z OKT3-antiCD28/RPMI T cells, n = 5, purple, straight line; 41BB.z OKT3-RetroNectin/LymphoONE T cells, n = 5, purple, dashed line). Tumor growth was followed by bioluminescence imaging. ( a ) Outline of the treatment schedule. ( b ) Representative images of JIMT-1.ffLuc-injected animals. ( c , left panel) Quantitative bioluminescence imaging data of JIMT-1.ffLuc xenografts (average total radiance = photons/s/cm 2 /sr; OKT3-antiCD28/RPMI HER2-CAR vs. OKT3-RetroNectin/LymphoONE HER2-CAR treatment: ** p < 0.01). ( c , right panel) Kaplan-Meier survival curve ( OKT3-antiCD28/RPMI HER2-CAR vs. OKT3-RetroNectin/LymphoONE HER2-CAR treatment: *** p < 0.001; OKT3-RetroNectin/LymphoONE NT vs. OKT3-RetroNectin/LymphoONE HER2-CAR treatment: *** p < 0.001). Histograms represent mean ± SD.

Article Snippet: Primary human T cells and CAR T cells were cultured in either RPMI (Roswell Park Memorial Institute, Buffalo, NY, USA) medium supplemented with 2 mmol/L GlutaMAX, 10% FBS, and antibiotics, or LymphoONE T-Cell Expansion Xeno-Free Medium (Takara Bio, Kusatsu, Japan) supplemented with 2 mM GlutaMAX, 10% FBS, and antibiotics.

Techniques: In Vivo, Injection, Imaging

Journal: Cancers

Article Title: Cytolytic Activity of CAR T Cells and Maintenance of Their CD4+ Subset Is Critical for Optimal Antitumor Activity in Preclinical Solid Tumor Models

doi: 10.3390/cancers13174301

Figure Lengend Snippet: EM-enriched CD28.z CAR.ffLuc T cells expand within one week post-infusion at tumor xenograft sites in vivo. Mice were injected s.c. with 3 × 10 6 JIMT-1 cells. Mice on day 14 (arrow) received a single i.v. dose of 2.5 × 10 6 NT.ffLuc T cells (NT.ffLuc OKT3-antiCD28/RPMI T cells, n = 4, grey, straight line; NT.ffLuc OKT3-RetroNectin/LymphoONE T cells, n = 3, grey, dashed line) or a single i.v. dose of 5 × 10 6 HER2-CAR.ffLuc T cells (CD28.z.ffLuc OKT3-antiCD28/RPMI T cells, n = 4, pink, straight line; CD28.z.ffLuc OKT3-RetroNectin/LymphoONE T cells, n = 4, pink, dashed line; 41BB.z.ffLuc OKT3-antiCD28/RPMI T cells, n = 4, purple, straight line; 41BB.z.ffLuc OKT3-RetroNectin/LymphoONE T cells, n = 4, purple, dashed line). Tumor infiltration was followed by bioluminescence imaging. ( a ) Outline of the treatment schedule. ( b ) Representative images of JIMT-1-injected animals. ( c ) Quantitative bioluminescence imaging data of HER2-CAR.ffLuc T cells infiltrating JIMT-1 xenografts (average total radiance = photons/s/cm 2 /sr; CD28.z.ffLuc OKT3-antiCD28/RPMI HER2-CAR vs. CD28.ffLuc OKT3-RetroNectin/LymphoONE HER2-CAR.ffLuc: *** p < 0.001) Histograms represent mean ± SD.

Article Snippet: Primary human T cells and CAR T cells were cultured in either RPMI (Roswell Park Memorial Institute, Buffalo, NY, USA) medium supplemented with 2 mmol/L GlutaMAX, 10% FBS, and antibiotics, or LymphoONE T-Cell Expansion Xeno-Free Medium (Takara Bio, Kusatsu, Japan) supplemented with 2 mM GlutaMAX, 10% FBS, and antibiotics.

Techniques: In Vivo, Injection, Imaging

Journal: Journal of Translational Medicine

Article Title: Large-scale expansion and characterization of CD3 + T-cells in the Quantum ® Cell Expansion System

doi: 10.1186/s12967-019-2001-5

Figure Lengend Snippet: Expansion of T-cells in the Quantum system

Article Snippet: PRIME-XV T-cell Expansion XSFM medium, a complete xeno-free, serum-free medium, was used for all T-cell expansions (FUJIFLM Irvine Scientific, Irvine, CA).

Techniques: Cell Culture, Concentration Assay

Journal: Nature Communications

Article Title: Hit-and-run programming of therapeutic cytoreagents using mRNA nanocarriers

doi: 10.1038/s41467-017-00505-8

Figure Lengend Snippet: mRNA nanoparticle transfection choreographs robust transgene expression by lymphocytes. a Primary T-cells were mixed with CD3-targeted polymeric nanoparticles (NPs) carrying Cy5-labeled mRNA. Confocal microscopy establishes that these particles are rapidly internalized from the cell surface. The images are representative of 15 randomly chosen fields. Scale bars , 2 μm. b Flow cytometry of preactivated PBMCs 24 h after incubation with CD3-targeted or isotype control antibody-targeted nanoparticles bearing eGFP-encoding mRNA. c Bar graph summarizing transfection efficiencies from three independent experiments conducted in duplicate. d , e Comparison of the effects electroporation and NP gene delivery have on cell expansion. Left panels show the workflow for transfection with NPs ( top ) and electroporation ( bottom ). Right panels show the -fold expansion of PBMC cultures from three independent donors treated with stimulatory beads on days 0 and 12. Matched cultures from each donor were not treated, or transfected using CD3/CD28-targeted NPs ( d , right ) or electroporation ( e , right ) on days 5 and 17. Every line represents one donor and each dot reflects the -fold T-cell expansion. Pairwise differences between groups were analyzed with the unpaired, two-tailed Student’s t test; n.s., non-significant; *, significant, n = 3). f Relative viability of NP-transfected and electroporated T-cells. Samples of 2 × 10 6 activated T-cells per condition were untreated, transfected with NPs, or electroporated. 18 h after treatment, cells were labeled with fluorescent dyes to assess viability. Results from three separate experiments conducted in duplicate are summarized in the bar graph shown in g . Statistical analysis between groups was performed using the unpaired, two-tailed Student’s t Test. * P < 0.0001

Article Snippet: For electroporation, 2 × 10 6 T-cells were washed twice with PBS containing 0.5% bovine serum albumin (BSA), resuspended in 100 μl of T-cell electroporation medium (Lonza) containing 3 μg of eGFP mRNA, transferred to an electroporation cuvette, and treated in a Nucleofector (Lonza) instrument using program T-20.

Techniques: Transfection, Expressing, Labeling, Confocal Microscopy, Flow Cytometry, Incubation, Control, Comparison, Electroporation, Two Tailed Test