Figure S1.
Imaging TCR and CAR on the surface of isolated T cells. (A and B) Blended view (A) and shaded binary (B) Imaris rendering of fixed anti-HER2 CAR T cell surface, as described in Fig. 1 A. Blended rendering shows section of 40 z slices. Scale bars are 2 μm. (C and D) Background intensity was subtracted from the sum of TCR intensity for a cell and divided by the approximate number of TCRs per cytotoxic T lymphocyte, which is at least ∼10,000 according to Blichfeldt et al. (1996) and Labrecque et al. (2001), in order to estimate the fluorescence intensity per TCR for each cell. That is, the estimated intensity per TCR was derived for each cell by dividing the sum of the background-subtracted surface intensity by 10,000. Patch regions were cropped and the intensity sum (minus background) for each patch was divided by the estimated intensity per TCR. 10 patches per cell were analyzed (C) and averaged for each cell (D). (E) Regions of interest (in addition to Fig. 1 B) that were used for the manual count of MV tip receptor occupancy in Fig. 1, C and D. MV tips are marked as being high for TCR (red arrow), CAR (green arrow), or both (yellow arrow). Scale bars = 2 μm. (F) Proportion of MV tips per region of interest that were marked as high for TCR, CAR, or both. 41 tips across 5 regions and 3 cells were scored. (G) Schematic of method used to define the cell membrane boundary used in radial intensity profiles. Outer boundary defined for each z-slice based off change in channel intensity. (H) Two additional examples of cells analyzed as described in Fig. 2, A–F. (I) Clusters are quantified for increasing thresholds of co-localization. Left: Number of TCR clusters with 0, 30, 50, or 70% overlap with CAR clusters. Right: Number of CAR clusters with 0, 30, 50, or 70% overlap with TCR clusters. 3 cells were scored, and data points for each cell are connected by lines. (J) Maximum intensity projection of TCR and CAR patches rendered in Imaris. For each patch, the intensity is defined by the integrated intensity for that patch. All patches are shown on the left. Patches which are ≥50% co-localized (i.e., 50% of the CAR patch overlaps with TCR, and vice versa) and <50% co-localized are shown in middle and left panels, respectively.

Imaging TCR and CAR on the surface of isolated T cells. (A and B) Blended view (A) and shaded binary (B) Imaris rendering of fixed anti-HER2 CAR T cell surface, as described in Fig. 1 A. Blended rendering shows section of 40 z slices. Scale bars are 2 μm. (C and D) Background intensity was subtracted from the sum of TCR intensity for a cell and divided by the approximate number of TCRs per cytotoxic T lymphocyte, which is at least ∼10,000 according to Blichfeldt et al. (1996) and Labrecque et al. (2001), in order to estimate the fluorescence intensity per TCR for each cell. That is, the estimated intensity per TCR was derived for each cell by dividing the sum of the background-subtracted surface intensity by 10,000. Patch regions were cropped and the intensity sum (minus background) for each patch was divided by the estimated intensity per TCR. 10 patches per cell were analyzed (C) and averaged for each cell (D). (E) Regions of interest (in addition to Fig. 1 B) that were used for the manual count of MV tip receptor occupancy in Fig. 1, C and D. MV tips are marked as being high for TCR (red arrow), CAR (green arrow), or both (yellow arrow). Scale bars = 2 μm. (F) Proportion of MV tips per region of interest that were marked as high for TCR, CAR, or both. 41 tips across 5 regions and 3 cells were scored. (G) Schematic of method used to define the cell membrane boundary used in radial intensity profiles. Outer boundary defined for each z-slice based off change in channel intensity. (H) Two additional examples of cells analyzed as described in Fig. 2, A–F. (I) Clusters are quantified for increasing thresholds of co-localization. Left: Number of TCR clusters with 0, 30, 50, or 70% overlap with CAR clusters. Right: Number of CAR clusters with 0, 30, 50, or 70% overlap with TCR clusters. 3 cells were scored, and data points for each cell are connected by lines. (J) Maximum intensity projection of TCR and CAR patches rendered in Imaris. For each patch, the intensity is defined by the integrated intensity for that patch. All patches are shown on the left. Patches which are ≥50% co-localized (i.e., 50% of the CAR patch overlaps with TCR, and vice versa) and <50% co-localized are shown in middle and left panels, respectively.

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