(A,B) Aminobisphosphonate-derived γδ T cells respond to aminobisphosphonates but not to bisphosphonates.

Risedronate was used to derive T-cell lines consisting of 80% to 90% γδ T cells (coexpressing Vγ2 and Vδ2 TCR chains; data not shown). These T-cell lines were rested for 3 weeks after the first stimulation and then restimulated for 24 hours with increasing concentrations of antigen, and collected supernatants were tested for IL-2 secretion by HT-2 cell proliferation and for IFN-γ secretion by enzyme-linked immunosorbent assay. (C) Antigenic aminobisphosphonates risedronate, alendronate, and pamidronate stimulated Vγ2Vδ2 T-cell expansion. Dose response is shown of aminobisphosphonates and of etidronate in stimulation of γδ T-cell expansion from 2 random leukopack PBMC preparations, as analyzed by flow cytometry. (D) Single-cell analysis of IFN-γ and TNF-α production by γδ T cells. Single-cell cytokine analysis performed by intracellular staining of IFN-γ and TNF-α using 3-color flow cytometric analyses is shown. A representative risedronate-derived line was restimulated with 10 μM concentration of aminobisphosphonates or etidronate for 17 hours. About 20% of cells in this line were monocytes or non-γδ T-cell lymphocytes; none of these stained positive for intracellular cytokines (data not shown). The number in the small box indicates the percent of γδ T cells that produced cytokines. (E) IL-2 release by TCR transfectants in response to aminobisphosphonates or etidronate. Vγ2Vδ2 and Vγ1Vδ1 TCR transfectants were stimulated with increasing concentrations of aminobisphosphonates or etidronate (upper panel) and with anti-CD3 mAb (OKT3) (lower panel) for 24 hours, and IL-2 release was measured by HT-2 cell proliferation.