Analysis of Immune Cells from Human Mammary Ductal Epithelial Organoids Reveals Vδ2⁺ T Cells That Efficiently Target Breast Carcinoma Cells in the Presence of Bisphosphonate

Characterization of immune cells from breast-derived organoids yields unique lymphocyte percentages compared with blood. A, gating strategy to delineate CD3⁺CD45⁺ T lymphocytes. B, quantification of CD3⁺CD45⁺ T lymphocytes. C, expression of the coreceptors CD8α⁺ and CD4⁺ by CD3⁺ T cells. D, quantification of CD8α⁺ expression by the CD3⁺ T cells. E, CD45RO^−/+ and CD27^−/+ (markers of T cell activation and differentiation) staining of CD8α⁺ T cells. F, quantification of CD8α⁺ T effector memory cells (CD45RO⁺CD27⁻). G, expression of CD103⁺ (an intraepithelial cell associated integrin) by cells of the CD8α⁺ gate. H, quantification of CD103⁺ CD8α⁺ T cells. ***, P < 0.0001 (Student t test).

Breast organoids contain specifically targetable lymphocytes that respond to an FDA-approved BP drug. A, flow cytometry analysis of an organoid preparation from tissue sample L1 (see Table 1) for γδ T cells and MAIT cells (Vα7.2 T cell receptor). B, quantification of T cell subsets from organoid preparations from the indicated tissue samples (see Table 1 for tissue donor information). Symbols under the dashed line were below the limit of detection. C, example of flow cytometry results showing Vδ2⁺ T cell expansion in the presence of 2.5 μmol/L BP compared with culture medium alone. Vα7.2⁺ MAIT cells were used as a control for nonresponsiveness to BP. D, quantification of Vδ2 T cell frequency from organoids compared to PBMCs as a positive control after 2 to 3 weeks of culture in the presence of BP. Five of 11 samples (45.5%) displayed Vδ2⁺ T cell expansion from the purified organoid fraction. Each symbol represents an independent expansion attempt using the indicated tissue samples. Dashed line indicates the threshold used to delineate expansion.

Breast organoid–derived Vδ2 γδ T cells produce IFNγ in response to a triple-negative breast carcinoma cell line pulsed with BP. A, Vδ2⁺ T cells were expanded in vitro from organoid preparations by exposure to BP. The T cells were coincubated with MDA-MB-468 breast carcinoma cells that were pulsed (right) or not pulsed (left) with BP, and intracellular IFNγ production was assessed by flow cytometry. Results shown are representative of two independent experiments. B, primary organoid-derived cells were coincubated with MDA-MB-468 cells that were pulsed (right) or not pulsed (left) with BP, and intracellular IFNγ production by Vδ2⁺ and Vδ2⁻ T cells was assessed by flow cytometric analysis. Numbers shown in the gates are the percentage of IFNγ–expressing cells from the Vδ2⁺ or Vδ2⁻ T cell populations. C, plot showing aggregated results for IFNγ production by primary Vδ2⁺ T cells from the indicated breast tissue samples. Normalized mean fluorescence intensity (MFI) for IFNγ was determined by dividing the IFNγ MFI of the Vδ2⁺ cells by the IFNγ MFI of the corresponding Vδ2⁻ T cells in the same sample. Each symbol represents an independent experiment to assess IFNγ production by T cells from the indicated breast tissue samples. The dashed line represents a normalization ratio of 1. n.s., not significant; *, P = 0.0121 (Mann–Whitney test; plus BP to ex vivo); *, P = 0.0262 (Mann–Whitney test; plus BP to no BP).

Cytotoxic functions by breast-derived Vδ2 γδ T cells in response to a triple-negative carcinoma cell line. A, Vδ2⁺ T cells were expanded in vitro from breast tissue preparations by exposure to BP. The T cells were coincubated with MDA-MB-468 breast carcinoma cells that were pulsed (right) or not pulsed (left) with BP, and cell surface expression of CD107a (LAMP-1, a marker of recent cytotoxic activity) was assessed by flow cytometry after 4 hours. B, MDA-MB-468 cells were pulsed (black squares) or not pulsed (gray squares) with BP, and coincubated with in vitro–expanded Vδ2⁺ T cells at the indicated effector:target ratios. The plot shows cytotoxicity of the target cells as assessed by cell-surface upregulation of Annexin V. Similar results were obtained in five independent experiments. C, primary organoid cells were coincubated with MDA-MB-468 cells that were pulsed (right) or not pulsed (left) with BP, and CD107a expression by T cells was assessed by flow cytometry. Numbers in the gates indicate the percentage of CD107a-expressing cells from the Vδ2⁺ or Vδ2⁻ T cell populations. The figure shows representative results from one out of seven independent experiments. D, plot showing aggregated results for cell-surface CD107a expression by primary T cells from the indicated breast tissue samples after exposure to MDA-MB-468 cells that were pulsed or not pulsed with BP, or by primary organoid-derived T cells that were not exposed to tumor cells ("ex vivo"). Each symbol represents an independent experiment to assess CD107a cell-surface expression by T cells from the indicated breast tissue samples. n.s., not significant; *, P = 0.0273; **, P = 0.0078 (Wilcoxon test).