Figure 2. Fas mediates geography-dependent off-target bystander killing of Ag⁻ tumor by Ag-specific CD8 T cells.

A, Cell counts of WT GFP⁺ (green) and WT mCherry⁺ (red) cells co-cultured with FACS purified tetramer⁺ (dark, GFP-specific) or tetramer⁻ (light, non-specific) activated CD8 T cells at indicated ratios; counts relative to beads and normalized to 0:1 condition; error bars presented as mean ± SEM; two-way ANOVA with Sidak correction for multiple comparisons, **p<0.01, ****p<0.0001 (n=4). B, Representative cleaved caspase 3 staining of GFP⁺ cells (middle row) and mCherry⁺ cells (bottom row) after co-culture with activated JEDI T cells in transwell configurations (top row, from left to right: GFP⁺ and mCherry⁺ in top chamber; GFP⁺ in top chamber / mCherry⁺ in bottom chamber; GFP⁺, mCherry⁺, and JEDI in top chamber; GFP⁺ and JEDI in top chamber / mCherry⁺ in bottom chamber; GFP⁺ and JEDI in top chamber / mCherry⁺ and JEDI in bottom chamber). C, Normalized cell counts of WT (white), B2m^−/− (gray), or Fas^−/− (black) mCherry⁺ cells when co-cultured with WT (left panel) or B2m^−/− (right panel) GFP⁺ cells in the presence of JEDI T cells; boxplots presented as minimum to maximum; two-way ANOVA with Sidak correction for multiple comparisons, ****p<0.0001 (n=4). D, Growth of mixed tumors with 50% WT GFP⁺ cells and 50% WT mCherry⁺ (black) or Fas^−/− mCherry⁺ (red) cells in Rag1^−/− mice adoptively transferred with tetramer-sorted GFP-specific CD8 T cells (arrow); percent change from maximum (day 6) to minimum (day 13) of each curve indicated; error bars presented as mean ± SEM; two-way ANOVA with Sidak correction for multiple comparisons, *p<0.05, **p<0.01 (n=9 mice/group; pooled from 2 independent experiments). Individual tumor curves in Supplemental Fig. S3D. E, Tiled confocal IF of tumors from mice bearing mixed primary GFP⁺/mCherry⁺ tumors (right column) and distant secondary mCherry⁺ tumors (left column) with either WT (top row) or Fas^−/− (bottom row) mCherry⁺ cells; tumors cryopreserved on days 6–8 after adoptive transfer of tetramer-sorted GFP-specific CD8 T cells (Supplementary Fig. S3F). F–G, Higher power images of primary GFP⁺/mCherry⁺ tumors with either WT (F) or Fas^−/− (G) mCherry⁺ cells from E (right column) with insets showing magnified single channel images of area outlined in yellow; arrows point to areas of active caspase 8 staining (bottom insets) and corresponding areas of mCherry staining (top insets). H, Quantification of percent area of mCherry staining that is colocalized with active caspase 8 staining from 37–39 fields of view as shown in F and G; unpaired two-tailed t test, ****p<0.0001 (n=3–5 mice/group, pooled from 3 independent experiments).