Profiling CAR T-cell Functionality

Viable CAR T cells were enriched using Ficoll. CD4⁺/CD8⁺ T-cell subsets were separated using anti-CD4 or anti-CD8 microbeads (Miltenyi Biotec) and stimulated with T3M4 and GPC1 KO T3M4 cells at a ratio of 1:2 for 20 h. A single-cell functional profile was determined as described previously^{28 (link),30 (link)}. Each sample’s polyfunctionality strength index (PSI) was computed using a prespecified formula^{28 (link)}, defined as the percentage of polyfunctional cells, multiplied by the mean fluorescence intensity (MFI) of the proteins secreted by those cells.

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Li N., Quan A., Li D., Pan J., Ren H., Hoeltzel G., de Val N., Ashworth D., Ni W., Zhou J., Mackay S., Hewitt S.M., Cachau R, & Ho M. (2023). The IgG4 hinge with CD28 transmembrane domain improves VHH-based CAR T cells targeting a membrane-distal epitope of GPC1 in pancreatic cancer. Nature Communications, 14, 1986.

Publication 2023

anti-CD4 or anti-CD8 microbeads

Cells Ficoll Fluorescence Microbeads Proteins T cell subsets T cells

Corresponding Organization :

Other organizations : National Institutes of Health, Center for Cancer Research, National Cancer Institute, IsoPlexis (United States), National Institute of Allergy and Infectious Diseases

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Variable analysis

independent variables

T-cell subsets (CD4+ and CD8+)

dependent variables

Polyfunctionality strength index (PSI) of T cells

control variables

T3M4 and GPC1 KO T3M4 cells used for stimulation at a ratio of 1:2
Stimulation duration of 20 hours

controls

Positive control: T3M4 cells
Negative control: GPC1 KO T3M4 cells

Annotations

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