In vitro HMBPP activation of Vγ2Vδ2 T cells in PBMCs, but not in the CD25⁺CD4⁺ cell culture containing Tregs, down-regulated IL-2–induced proliferation/expansion of CD4⁺CD25⁺Foxp3⁺ regulatory T cells. (A) Flow cytometry histograms showed changes in percentages of CD4⁺CD25⁺Foxp3⁺ T cells for one representative macaque (7404) after the 8-day culture with IL-2 or IL-2 + HMBPP (top panels were CD4 gated). Panels B and C show percentages of Tregs and Vγ2Vδ2 T cells, respectively, after the 8-day culture with IL-2 only or IL-2 + HMBPP. Data were mean values with SEM derived from 3 independent experiments using PBMCs from 7 naive cynomolgus monkeys. **P < .01; *P < .05 for the comparisons. (D,E) CD4⁺CD25⁺ T cells and activated Vδ2 T cells proliferated mutually in the presence of IL-2 in the culture without APCs and other immune cells. Purified Vδ2⁺ T cells (0 × 10⁵, 1 × 10⁵, 2 × 10⁵, or 4 × 10⁵) were mixed with 10⁵ cells/well of CD4⁺CD25⁺ T cells, labeled with CFSE, and then cultured with 20 U/mL IL-2 for 7 days. Proliferation was analyzed by flow cytometry to determine the dilution of CFSE fluorescence intensity: the number of CFSE^dim cells divided by the number of CFSE⁺ cells. (D) Flow histograms of one representative macaque (7716). (E) Percentage rates of proliferation of CD4⁺CD25⁺ T cells and Vδ2 T cells after the 7-day culture. Data were mean values with SEM from 3 independent experiments using PBMCs from 6 naive macaques. **P < .01; *P < .05 for differences between CD4⁺CD25⁺ T cells only and these cells plus Vδ2 T cells. Purified CD25⁺CD25⁺ T cells were shown to inhibit antigen-specific T cells (data not shown).