Anti–IFN-γ, but not anti–TGF-β or anti–IL-4, neutralizing antibody reduced the ability of HMBPP-activated Vγ2Vδ2 T cells to down-regulate the IL-2–induced expansion of CD4⁺CD25⁺Foxp3⁺ T cells. (A) Flow histograms from one representative monkey (7741) show that IL-2–induced expansion of Tregs was inhibited by HMBPP + IL-2, but the inhibition was reversed significantly by adding neutralizing anti–IFN-γ mAb (1 or 3 μg/mL) to HMBPP + IL-2 in PBMC culture. No effect was seen when adding neutralizing anti–TGF-β mAb, or neutralizing anti–IL-4 mAb to the HMBPP + IL-2 culture. (B,C) Numbers of CD4⁺CD25⁺Foxp3⁺ T cells and Vγ2Vδ2 T cells in PBMC cultures treated with medium alone, IL-2, HMBPP + IL-2, HMBPP + IL-2 + anti–TGF-β mAb, HMBPP + IL-2 + anti–IL-4 mAb, and HMBPP + IL-2 + anti–IFN-γ mAb. Data were mean values with error bars of SEM from PBMCs of 3 monkeys in 2 independent experiments. (D) Concentrations of IFN-γ protein in PBMC cultures treated with medium alone, IL-2, HMBPP + IL-2, HMBPP + IL-2 + anti–TGF-β mAb, HMBPP + IL-2 + anti–IL-4 mAb, and HMBPP + IL-2 + anti–IFN-γ mAb. **P < .01; *P < .05.

Activation of Vγ2Vδ2 T cells by Picostim/IL-2 treatment not only down-regulated the IL-2–induced expansion of Tregs, but also reversed subsequent Treg-associated suppression of Ag-specific antimicrobial T-cell responses in mycobacterial infection. (A) Intracellular cytokine staining after antigen stimulation showed that numbers of HMBPP-specific Perforin⁺ (left panel) and IFNγ⁺ (middle panel) Vγ2Vδ2 T cells were significantly higher in PBMCs of BCG-infected IL-2/Picostim–treated monkeys than those in BCG-infected IL-2–treated macaques. Data were mean values with SEM derived from PBMCs of 6 monkeys in each group. *P < .05 for differences in perforin⁺ cells between BCG + IL-2 and BCG + IL-2/Picostim groups at weeks 2 through 4; P < .05 for differences in IFNSγ⁺ cells between BCG + IL-2 and BCG + IL-2/Picostim groups at weeks 2 and 4. Similarly, numbers of PPD-specific IFN⁺ CD4 T cells were higher in PBMCs of BCG-infected IL-2/Picostim–treated monkeys than those in BCG-infected IL-2–treated macaques. *P < .05 for differences in IFNγ⁺ between BCG + IL-2 and BCG + IL-2/Picostim groups at weeks 3 and 4. (B) BCG appeared to be more isolable in the blood of BCG + IL-2 group than of BCG + IL-2/Picostim–treated group at week 1. Data were mean values with SEM derived from blood of 6 monkeys per group.

A 5-day IL-2 administration regimen induced an apparent expansion of CD4⁺CD25⁺Foxp3⁺ T cells (Tregs) in PBMCs; adding Picostim to the IL-2 treatment regimen induced Vγ2Vδ2 T-cell expansion and down-regulated IL-2–induced increases in numbers of Tregs in BCG-infected macaques. (A) The top panel of flow cytometry histograms showed that one representative macaque (7285) exhibited increased numbers of CD25⁺Foxp3⁺CD4 T cells after the IL-2 treatment (on the left), with subtle changes in Vγ2Vδ2 T cells (on the right). The bottom panel of flow cytometry histograms showed that the other representative monkey (7287) displayed reduced numbers of CD25⁺Foxp3⁺CD4 T cells after the IL-2 + Picostim treatment compared with the IL-2–treated monkey, with marked increases in Vγ2Vδ2 T cells (on the right). CD25⁺Foxp3⁺ T cells were gated on CD4. Note that the percentages of CD25⁺Foxp3⁺ Tregs in CD3⁺ T cells were 4% on day 5 after the IL-2 + Picostim treatment. Also see total numbers of these T cells in panel B. (B) Changes in percentage and absolute numbers of CD4⁺CD25⁺Foxp3⁺ T regulatory cells in PBMCs of 3 groups of monkeys (BCG; BCG + IL-2; BCG + IL-2 + Picostim) over time after treatment/infection. Shown are the mean values with SEM from 6 monkeys for each group. **P < .01; *P < .05 for differences between BCG + IL-2 and BCG groups and between BCG + IL-2 and BCG + IL-2. (C) Changes in percentage and absolute numbers of Vγ2Vδ2 T cells in PBMCs of 3 groups of monkeys (BCG; BCG + IL-2; BCG + IL-2 + Picostim) over time after treatment/infection. **P < .01; *P < .05 for differences between BCG + IL-2 and BCG groups, and between BCG + IL-2 + Picostim and BCG groups or BCG + IL-2 + Picostim and BCG + IL-2 groups. Note that IL-2–induced expansion of Tregs (B) led to subsequent suppression of BCG-induced expansion of Vγ2Vδ2 T cells at days 21 to 42 (*P < .05 for differences between BCG + IL-2 and BCG groups as well as BCG + IL-2 and BCG + IL-2 + Picostim groups at days 21 and 28).

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).