Journal: Journal of Immunotherapy (Hagerstown, Md. : 1997)
Article Title: Development of Engineered CAR T Cells Targeting Tumor-Associated Glycoforms of MUC1 for the Treatment of Intrahepatic Cholangiocarcinoma
doi: 10.1097/CJI.0000000000000460
Figure Lengend Snippet: In vitro cytotoxicity of 5E5 chimeric antigen receptor (CAR) T cells. A, Schematic representation of 5E5 CAR. The 5E5 CAR was comprised of a scFv, human CD8 hinge, and transmembrane region derived from human CD8 transmembrane region, as well as the intracellular signaling domains of human 4-1BB and CD3ζ. B, Flow cytometric analysis of the expression of 5E5 CAR in transduced T lymphocytes. Mock T and 5E5 CAR T cells were stained with biotinylated goat antimouse immunoglobulin G and streptavidin-PE. C, Mock or 5E5 CAR T cells were incubated with HuCCT1-WT or HuCCT1-KO at different effector: target (E:T) ratios for 24 hours. Then the production of interferon (IFN)-γ and tumor necrosis factor (TNF)-α was determined by enzyme-linked immunosorbent assay. Data representative of three independent experiments were shown. D, In vitro cytotoxic activities of 5E5 CAR T cells. The Mock or 5E5 CAR-modified T cells were incubated with CFSE-labeled HuCCT1-WT or HuCCT1-KO cell lines at an E:T ratio of 1:1 for 18 hours. Flow cytometric analysis of target cells stained with CFSE and APC-labeled Annexin V. E, Mock or 5E5 CAR T cells were incubated with HuCCT1-WT or HuCCT1-KO at different E:T ratios for 18 hours. Specific lysis of target cells was measured by performing the cell counting kit-8 (CCK-8) assay on the remaining live target cells. The error bars indicated the SD, and an unpaired two-tailed Student’s t test was used for comparisons to determine statistical significance (* P <0.05, *** P <0.001). APC indicates allophycocyanin; CFSE, carboxyfluorescein diacetate succinimidyl ester.
Article Snippet: In brief, 1×10 6 cells were suspended in cold PBS and incubated with 5E5 antibody or biotin-streptavidin-conjugated goat antimouse immunoglobulin (Ig)G (Fab) (Jackson Immunoresearch, 115-065-072) for one hour at 4°C.
Techniques: In Vitro, Derivative Assay, Expressing, Staining, Incubation, Enzyme-linked Immunosorbent Assay, Modification, Labeling, Lysis, Cell Counting, CCK-8 Assay, Two Tailed Test
SouthernBiotech is a verified supplier 
