Figure 6.

Vγ2Vδ2 T cells expanded by HMBPP + IL-12 differentiate into polyfunctional effector cells expressing antimicrobial IFN-γ, TNF-α, GM-CSF, and tri-CTL cytokines. (A) Vγ2Vδ2 T cells expanded by HMBPP + IL-12 expressed higher level of TBX21 than those expanded by HMBPP/IL-2. PBMC were cultured with HMBPP + IL-12 (white bars) or HMBPP + IL-2 (black bars) for 7 days, and expanded Vγ2Vδ2 T cells were purified. Then, enriched Vγ2Vδ2 T cells were used for RNA isolation and determination of the expression levels of TBX21 and FOXP3. Cells from HMBPP + IL-2 treatment served as control setting. Shown are mean ± SEM of three independent experiments pooled from 15 healthy controls, ^****p < 0.0001, t-test. (B) Pooled flow-cytometry data (12 healthy controls) show the frequencies of T-bet⁺ cells (left) and mean fluorescence index (MFI) expression of Foxp3 (right) in gated Vγ2Vδ2 T cells expanded by HMBPP + IL-12 or HMBPP + IL-2. Each dot represents one healthy control. ^***p < 0.001, paired t-test. (C) Percentage numbers of Vγ2Vδ2 T cells expanded by HMBPP + IL-12 or HMBPP + IL-2 could produce CD107a, IFN-γ, TNF-α, and GM-CSF in 15 combinations in response to HMBPP stimulation. Using Boolean analysis, the bar graph shows percentages of individual multi-functional effector subsets for Vγ2Vδ2 T cells expanded by HMBPP + IL-12 (white bars) and HMBPP + IL-2 (black bars). Gating was on individual live lymphocytes, CD3⁺ T, then Vγ2⁺Vδ2⁺ T cells and then those cytokine markers. Shown are mean ± SEM of three independent experiments pooled from 15 healthy controls, ^****p < 0.0001, ^***p < 0.001, t-test. (D) Bar graph shows fold changes in expression levels of GZMA, GZMB, GNLY, and PRF in HMBPP + IL-12-expanded Vγ2Vδ2 T cells after HMBPP or media stimulation. Data are from four independent experiments pooled from 15 healthy controls. ^****p < 0.0001, ^***p < 0.001, ^**p < 0.01, t-test. Representative flow cytometric histograms are in Supplementary Figure 5A.