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Fig. 3 | Journal of Hematology & Oncology

Fig. 3

From: Incorporation of a hinge domain improves the expansion of chimeric antigen receptor T cells

Fig. 3

A hinge enhances the migratory capacity of CAR T cells. Cytotoxicity of (a) 19.28z T, 19-H.28z T, and control GFP T cells after co-culture with CD19+ cell line (NALM6-GL) for 24 h, (b) Meso.28z T, Meso-H.28z T, and control GFP T cells after co-culture with mesothelin + cell line (A549-GL) for 24 h. E:T ratios are the ratios of the absolute number of CAR T cells to the target cells. The GFP percentages of the CAR T cells were equalized using non-transduced T cells from the same donor. n = 3 replicates per point; the data are representative of independent experiments verified with cells from over three individual healthy human donors. IL2 production of (c) 19.28z T, 19-H.28z T, and control GFP T cells after co-culture with CD19+ cell line (NALM6-GL) for 24 h, (d) Meso.28z T, Meso-H.28z T, and control GFP T cells after co-culture with mesothelin + cell line (A549-GL) for 24 h. IFN-γproduction of (e) 19.28z T, 19-H.28z T, and control GFP T cells after co-culture with CD19+ cell line (NALM6-GL) for 24 h, (f) Meso.28z T, Meso-H.28z T, and control GFP T cells after co-culture with mesothelin + cell line (A549-GL) for 24 h. CAR T cells were co-cultured with the targeted tumor cells at a 1:1 E: T ratio. n = 3 replicates per point; the data are representative of independent experiments verified with cells from over three individual healthy human donors. Error bars denote the SEM, and the results were compared through an unpaired t test. *P < 0.05, **P < 0.01, and ***P < 0.001. Transwell cell migration assay indicated an improved capacity of (g) 19-H.28z T cells, (h) Meso-H.28z T cells to transmigrate Matrigel. For the 19.28z T and 19-H.28z T cells, Nalm6 cell lysates were used as a chemoattractant in the lower chamber, while for the Meso.28z T and Meso-H.28z T cells, A549 cell lysates were used as a chemoattractant in the lower chamber. The T cells were cultured in an insert coated with Matrigel for 24 h, and the cells that transmigrated to the lower chamber were counted by flow cytometry. The percentage of migration was calculated as follows: (CAR T cells migrating through the Matrigel chamber membrane/total CAR T cells in insert membrane before assay begin) × 100. n = 3 replicates per point; the data are representative of independent experiments verified with cells from over three individual healthy human donors. Error bars denote the SEM, and the results were compared through an unpaired t test. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001

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