Fig. 1

Multiplexing single cell measurement of CAR-T cells in microchambers. a Schematic outline of CD19TL19 CAR-T cell generation, sorting and stimulation. The CD19-BB-z transgene lentiviral vector was used for CAR-T cell generation. CAR-T cells were sorted and then stimulated by anti-CAR beads prior to the SCBC assay. b The validated 16-plex panel including 4 groups of cytokines: effector, stimulatory, regulator and inflammatory. c Major work flow for single-cell functional proteomic analysis. (i) Schematic depiction of a microchamber fabricated in PDMS used for isolating single cells and assaying a panel of proteins/cytokines secreted from the entrapped cell with an antibody barcode array patterned on the glass slide. Each device has 12,000 microchambers. (ii) Motorized miscopy allows for automated imaging of the entire microchamber PDMS device for locating and counting T cells in microchambers. Protein secretion profile is obtained by quantifying the fluorescence signals corresponding to single-cell secretions in each microchamber. Overlay of these two data sets allows for the identification of single-cell protein secretion profiles. (iii) Quantitative analysis, statistics and advanced informatics (e.g., CytoSpeak package) were applied in this project to investigate the effector function (cytokine) landscape of single CAR-T cells. d A representative measurement of IFN-γ in supernatants from CAR-T cells stimulated by either IgG beads or anti-CAR beads by ELISA