Comparison of different bisphosphonates to stimulate γδ T cells.
(A) Capacity of different bisphosphonates to stimulate γδ T cells. Primary PBMC of healthy donors were incubated with increasing concentrations of different bisphosphonates and IPP as a positive control and with low doses of IL-2 (10 U/mL). Percentage of γδ T cells was determined by FACS analysis using anti-CD3 and anti-γδ TCR mAb after 7 days of culture. Results are shown as mean ± SD of triplicate cultures in one representative donor. Similar dose–response curves were observed in 5 healthy donors. Maximum γδ T-cell increase in aminobisphosphonate-stimulated cultures of different donors ranged from 20% to 70%. PBMC cultures with bisphosphonates or IPP alone (without exogenous IL-2) or cultures with IL-2 alone exhibited no significant increase of the γδ T-cell proportion (always less than 10%). • = IPP; ▴ = pamidronate; □ = etidronate; ◊ = clodranate; ⊞ = alendronate; and ⊕ = ibandronate. (B) Proliferation of γδ T cells stimulated by aminobisphosphonates. Primary PBMC were incubated with increasing concentrations of the non-aminobisphosphonate clodronate or the aminobisphosphonate pamidronate in the presence or absence of IL-2 (10 U/mL). Absolute numbers of γδ T cells were calculated on day 7 by counting the absolute number of viable cells per well and measuring the percentage of γδ T cells by FACS analysis. Control cultures revealed the following absolute γδ T-cell numbers: medium alone = 0.44 × 103; medium + IL-2 = 0.90 × 103; IPP (4 μmol/L) alone = 0.33 × 103; IPP (4 μmol/L) + IL-2 = 45 × 103. Results are shown as mean values of triplicate cultures in 1 donor and are representative of similar experiments with 3 normal donors (range of γδ T-cell expansion in pamidronate/IL-2 cultures between 50- and 100-fold compared with medium or clodronate/IL-2 cultures). ◊ = clodranate; ⧫ = clodranate + IL-2; ▵ = pamidronate; and ▴ = pamidronate + IL-2.
