Figure S3.
CAR T cell calcium flux and close contact fluorescence. (A) Anti-HER2-BV605 staining was performed on the human breast cancer cell lines MCF7 and SKBR3, plus murine MC38 cells as a HER2-negative control. Histogram overlay shows low and high HER2 expression for MCF7 and SKBR3, respectively. (B) Lipid bilayers with varying HER2 densities were built on 5 μm microspheres as standards for comparison to high-HER2 expressing SKBR3 and low-HER2 expressing MCF7s. Representative histogram from two independent experiments. (C) Fura-2 ratio images showing calcium flux (yellow) with tracks for interactions of CAR T cells with HER2-bilayers across antigen density and CAR affinity. Control well with no HER2 is shown on left. Images shown are at 3 min following addition of T cells. Scale bars are 7 μm. (D) Fura-2 ratio imaging shows calcium flux across affinity and HER2 density, with LA CAR T cells outperforming HA CAR T cells on high-HER2 loaded bilayers. Top: Maximum Fura-2 ratio for cells was quantified at 5.5 min following addition of T cells. Error bars represent SD (n = 37, 23, 21, 33, 66 cells per condition from left to right, respectively). Analyses are unpaired t tests. Bottom: Percentage of cells fluxing (defined as maximum Fura-2 ratio >100) for each condition was quantified at 5.5 min following addition of T cells. (E) Schematic of original anti-HER2 CAR, including two cysteines in the CD8 hinge region that form disulfide bond, and monomeric CAR made by mutation of those cysteines to serine (Hennecke and Cosson, 1993). (F) The average close contact CAR-mEmerald fluorescence intensity was not significantly different between monomers and dimers. Data is shown for at least 5 cells per condition across three experiments (n = 7, 7, 5, 13 cells per group from left to right, respectively, and ≥505 close contacts were averaged per cell). Error bars represent SD, analyses shown are unpaired t tests. (G) Average close contact CAR-mEmerald fluorescence intensity was plotted against persistence time following color schema in Q. Linear trend lines were added for each CAR type (monomers dashed).

CAR T cell calcium flux and close contact fluorescence. (A) Anti-HER2-BV605 staining was performed on the human breast cancer cell lines MCF7 and SKBR3, plus murine MC38 cells as a HER2-negative control. Histogram overlay shows low and high HER2 expression for MCF7 and SKBR3, respectively. (B) Lipid bilayers with varying HER2 densities were built on 5 μm microspheres as standards for comparison to high-HER2 expressing SKBR3 and low-HER2 expressing MCF7s. Representative histogram from two independent experiments. (C) Fura-2 ratio images showing calcium flux (yellow) with tracks for interactions of CAR T cells with HER2-bilayers across antigen density and CAR affinity. Control well with no HER2 is shown on left. Images shown are at 3 min following addition of T cells. Scale bars are 7 μm. (D) Fura-2 ratio imaging shows calcium flux across affinity and HER2 density, with LA CAR T cells outperforming HA CAR T cells on high-HER2 loaded bilayers. Top: Maximum Fura-2 ratio for cells was quantified at 5.5 min following addition of T cells. Error bars represent SD (n = 37, 23, 21, 33, 66 cells per condition from left to right, respectively). Analyses are unpaired t tests. Bottom: Percentage of cells fluxing (defined as maximum Fura-2 ratio >100) for each condition was quantified at 5.5 min following addition of T cells. (E) Schematic of original anti-HER2 CAR, including two cysteines in the CD8 hinge region that form disulfide bond, and monomeric CAR made by mutation of those cysteines to serine (Hennecke and Cosson, 1993). (F) The average close contact CAR-mEmerald fluorescence intensity was not significantly different between monomers and dimers. Data is shown for at least 5 cells per condition across three experiments (n = 7, 7, 5, 13 cells per group from left to right, respectively, and ≥505 close contacts were averaged per cell). Error bars represent SD, analyses shown are unpaired t tests. (G) Average close contact CAR-mEmerald fluorescence intensity was plotted against persistence time following color schema in Q. Linear trend lines were added for each CAR type (monomers dashed).

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