FIGURE 1.

Differential effector functions of BCG- and IPP-expanded γ₉δ₂ T cells. A, Both BCG and IPP expand Vγ₉⁺Vδ₂⁺ γδ T cells. After 1 week of rest in medium alone or expansion with either BCG or IPP, γδ T cells present were identified by staining with anti-CD3 and either a pan-γδ TCR-specific mAb, or Vγ₉and Vδ₂ specific mAb. Virtually all γδ T cells expanded with either BCG or IPP expressed both Vγ₉ and Vδ₂. None of the expanded γδ T cells expressed detectable Vγ₁ or Vδ₁ chains (data not shown). B, BCG- but not IPP-expanded PBMC inhibit intracellular mycobacteria. PBMC were cultured in vitro with BCG, IPP + IL-2, or in medium alone for 7 days, and then co-cultured with autologous BCG-infected macrophages. BCG viability was determined 3 days later by H³-Uridine incorporation. PBMC expanded with BCG could inhibit intracellular mycobacterial growth (n=12/group; *p<0.005). PBMC expanded with IPP did not inhibit intracellular BCG (NS, p = 0.81 by Wilcoxon matched-pairs tests). C, Purified BCG-expanded γ₉δ₂ T cells inhibit intracellular mycobacteria. BCG expanded and purified γδ T cells (purity > 98% γδ-TCR⁺, CD3⁺ T cells), or total BCG-expanded PBMC, were co-cultured with infected macrophages at an effector:target ratio of 10:1. Both total BCG-expanded PBMC and purified BCG-expanded γδ T cells significantly inhibited intracellular mycobacterial growth compared with cultures containing medium rested cells alone (n=9/group; *p<0.01 by Wilcoxon matched pairs test), and the inhibitory effects of purified γδ T cells were significantly greater than the effects of total BCG-expanded PBMC (n=9/group; **p=0.011). D, Both BCG- and IPP-expanded γδ T cells express cytolytic effector functions. γδ T cells were purified from PBMC after 7 days of expansion with BCG or IPP+IL-2. Cr⁵¹-labeled Daudi cells and purified γδ T cells were cultured at various Effector:Target ratios for 4 hours at 37°C. Supernatants were harvested and percent lysis calculated as described. E, Mycobacteria and phosphoantigen stimulation induce similar levels of key effector molecules. PBMC from 2 PPD+ volunteers (Vol#1 and Vol#2) were rested in medium (open histogram with thin line), stimulated with 5 μg/mL mycobacterial lysate (open histogram with thick line), or stimulated with 250 pM HMB-PP (filled histogram) for 7 days. The levels of intracellular perforin, granzyme A, granulysin and IFN-γ were assessed by intracellular cytokine staining. Results shown are gated on CD3⁺ Vδ₂⁺ T cells.