Figure 4 from Breast Cancer Stem Cell–Derived Tumors Escape from γδ T-cell Immunosurveillance In Vivo by Modulating γδ T-cell Ligands

γδ T cells fail to control BCSC5 xenografts in NOD SCID mice. A, Kaplan–Meier plot (left) of BCSC5 xenograft-bearing mice upon treatment with γδ T cells (blue), γδ T cells expressing MMP14 (red) or vehicle control (black; n = 6–7 mice per group). Differences were not statistically significant, log-rank test (Mantel-Cox). BCSC5 tumor growth curves (right) for individual mice after treatment start. B, γδ T cell-mediated degranulation (left) and cytotoxicity (right) of BCSC5 culture cells or xenograft-derived tumor cells (xeno). Xenograft-bearing mice were treated with γδ T cells, γδ T cells expressing MMP14 or vehicle. For the degranulation assay, γδ T cells were cocultured with the respective tumor cells for 3 hours. The percentage of CD107a⁺ cells of Vδ2⁺-gated cells is shown (means ± SEM). Four to six tumors were analyzed per group and each tumor was tested with 2 healthy donors of γδ T cells. For in vitro killing of ⁵¹Cr-labeled tumor cells, γδ T cells were cocultured with the target cells for 5 hours at an E:T ratio of 30:1. Results from three independent experiments with a total of 3 healthy donors of γδ T cells and three tumors per group were pooled (means ± SEM). One-way ANOVA followed by Dunnett post hoc test comparing xenograft with culture cells. C, Flow cytometry-based analysis of PD-L1 and PD-L2 expression levels in xenograft-derived EpCAM⁺ tumor cells (median, minimum to maximum). Mean fluorescence intensity (MFI) of 9–14 tumors per group is shown. Kruskal–Wallis test followed by Dunn post hoc test comparing xenograft with culture cells. D, Kaplan–Meier plots of NOD SCID mice upon treatment with vehicle control (black), γδ T cells (blue) or γδ T cells expressing MMP14 (red) in combination with an anti–PD-1 (nivolumab) or isotype control antibody (n = 5–6 per group). Treatment start was defined for each mouse individually when the first tumor reached a volume of at least 4 mm³. A total of 5 × 10⁶ γδ T cells were injected intravenously three times per week. In addition, mice received 0.6 × 10⁶ IU IL2 (Proleukin S) on the day of treatment start and every 21 days until the end of the experiment. The end of the experiment was defined by a tumor volume of 800 mm³. No significant differences were obtained, log-rank (Mantel-Cox) test. E, BCSC5 tumor growth curves for individual mice upon treatment with γδ T cells (blue), γδ T cells expressing MMP14 (red) or vehicle control (black) in combination with anti–PD-1 or isotype control antibody (n = 5–6 per group). *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.