Self-Maintaining CD103⁺ Cancer-Specific T Cells Are Highly Energetic with Rapid Cytotoxic and Effector Responses

Tumor antigen sensitivity of CD103⁺ cancer-specific T cells. Proportion of CD103⁺ IFNγ⁺ T cells (A) and CD107a⁺ T cells (B) with either anti-CD103 blocking treatment, isotype control treatment, or no treatment following coculture with HCT116 cells at five different concentrations of antigen. SSX-2 (left) and NY-ESO-1 (right) CD103⁻ T cells with no treatment also shown (N, number of repeats = 3). C, Proportion of lysed HCT116 cancer cells following coculture with either CD103⁺ T cells left untreated or treated with anti-CD103 or isotype at five different antigen concentrations. CD103⁻ T cells with no treatment also shown (N, number of repeats = 3). D, Proportion of CD103⁺CD107a⁺ SSX-2–specific T cells following coculture with either E-Cadherin⁺ HCT116 cells or E-Cadherin⁻ THP-1 cells at five different concentrations of antigen (N, number of repeats = 3). E, Proportion of cancer cell death following coculture with either E-Cadherin⁺ HCT116 cells or E-Cadherin⁻ THP-1 cells at five different concentrations of antigen. A–C, Data, median ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001. P values were calculated using two-way ANOVA with Tukey post hoc analysis.

Mechanism of CD103⁺ cancer-specific T-cell cytotoxicity. A, Confocal imaging time lapse of 1 μg SSX-2 antigen–loaded HCT116 cancer cells cocultured either CD103⁺ (top) or CD103⁻ (bottom) SSX-2–specific T cells labeled with Fluo4-AM for calcium staining. Close-up images showing brightfield (top), Fluo4-AM stain (middle), and merged (bottom). Fluo4-AM calcium staining shown in red. Scale bar, 10 μm. Blue dotted box indicates zoomed imaged area at a magnification of 25×. B, Amount of time (in seconds) required for the first instance of 1 μg SSX-2 antigen–loaded HCT116 cancer cells cocultured with either CD103⁺ (top) or CD103⁻ (bottom) SSX-2–specific T cells. N₁, number of events collected for CD103⁺ T cells: 29; N₂, number of events collected for CD103⁻ T cells: 29 (N, number of repeats = 3; at 95th percentile). C, Confocal imaging time-lapse 1 μg SSX-2 antigen–loaded HCT116 cancer cells cocultured with either CD103⁺ (top) or CD103⁻ (bottom) SSX-2–specific T cells labeled with Annexin V. Close-up images showing brightfield (top), Annexin V (middle), and merged (bottom) at 3, 4, 5, 6, 9, and 12 hours after coculture. Annexin V staining shown in blue. Scale bar, 10 μm. Blue dotted box indicates zoomed imaged area at a magnification of 25×. D, Normalized Annexin V intensity representing HCT116 cell death across a 12-hour time period following 1 μg SSX-2 antigen–loaded HCT116, and cocultured with either CD103⁺ (top) or CD103⁻ (bottom) SSX-2–specific T cells. A close-up detailing 2- to 8-hour coculture (N, number of repeats = 3) is also shown. E, Percentage of HCT116 cell death across a 12-hour time period following 1 μg SSX-2 antigen–loaded HCT116, and cocultured with either CD103⁺ (top) or CD103⁻ (bottom) SSX-2–specific T cells (N, number of repeats = 3). Data, median ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001. P values were calculated using either paired Student t test with Wilcoxon adjustments, one-way ANOVA, or two-way ANOVA with Tukey post hoc analysis.

Metabolic activities of CD103⁺ cancer-specific T cells. A, The ECAR of antigen-stimulated CD103⁺ or CD103⁻ T cells across an 80-minute period. SSX-2 (left) and NY-ESO-1 (right; N, number of repeats = 3). Injection of glucose, oligomycin, and 2-DG into cells is indicated. The ECAR of basal glycolytic capacity (B) and the maximal glycolytic capacity (C) of antigen-stimulated CD103⁺ or CD103⁻ T cells (N, number of repeats = 3). D, The OCR of antigen-stimulated CD103⁺ or CD103⁻ T cells across an 80-minute period. SSX-2 (left) and NY-ESO-1 (right; N, number of repeats = 3). Injection of oligomycin, FCCP, and rotenone/antimycin A into cells is indicated. The OCR at basal respiration stage (E) and the spare respiratory capacity (F) of antigen-stimulated CD103⁺ or CD103⁻ T cells (N, number of repeats = 3). Data, median ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001. P values were calculated using either paired Student t test with Wilcoxon adjustments, one-way ANOVA, or two-way ANOVA with Tukey post hoc analysis.

CD103 expression on T cells enables faster migration and homing. A, The number of CD103⁺ or CD103⁻ SSX-2–specific T cells that migrated across a transwell membrane toward HCT116 cells, over a time period of 2 hours, at an E:T ratio of 1:4. SSX-2–specific T-cell clones shown on the left and NY-ESO-1–specific T clones shown on the right (N, number of repeats = 3). B, The number of CD103⁺ or CD103⁻ T cells that migrated across a transwell membrane after 60 minutes with or without anti-CD103 blocking treatment. SSX-2–specific T-cell clones shown on the left and NY-ESO-1–specific T clones shown on the right (N, number of repeats = 3). Data, median ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant. P values were calculated using either one-way or two-way ANOVA with Tukey post hoc analysis.

CD103⁺ CD8⁺ TILs localize on E-Cadherin–rich tumors. A, Proportion of CD103⁺CD8⁺ T cells (left) or CD103⁻CD8⁺ T cells (right) present either within, clustering around, or distal from E-cadherin⁺ cells (N, number of patients = 5; at 95th percentile). B, Density (by μm²) of CD103⁺CD8⁺ T cells (left) or CD103⁻CD8⁺ stained cells (right) present either within, clustering around, or distal from E-cadherin⁺ cells (N, number of patients = 5). Data, median ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant. P values were calculated using one-way ANOVA with Tukey post hoc analysis.