(E)-4-hydroxy-3-methyl-but-2 enyl pyrophosphate-stimulated Vgamma9Vdelta2 T cells possess T helper type 1-promoting adjuvant activity for human monocyte-derived dendritic cells

Abstract

Vgamma9Vdelta2 T cells respond to pyrophosphate antigens and display potent antitumour activity in vitro. We have investigated the potential of the most potent phosphoantigen known to activate Vgamma9Vdelta2 T cells, (E)-4-hydroxy-3-methyl-but-2 enyl pyrophosphate (HMB-PP), as an adjuvant for dendritic cell (DC)-based vaccines. A single stimulation of peripheral blood mononuclear cells with HMB-PP and IL-2 was sufficient to generate lines of effector memory Vgamma9Vdelta2 T cells that retained their cytolytic and cytokine secretion activities. These cells induced differentiation of DC into semi-mature antigen-presenting cells expressing CD86, CD11c, CD54, HLA-DR, CD83 and CD40, which secreted low levels of bioactive IL-12 but no IL-10. Vgamma9Vdelta2 T cells also strongly costimulated IL-12 release but inhibited IL-10 production by lipopolysaccharide (LPS)-stimulated DC. When substituted for Vgamma9Vdelta2 T cells, IFN-gamma did not induce full DC maturation but it augmented IL-12 and inhibited IL-10 release by LPS-stimulated DC, in a manner similar to HMB-PP-activated Vgamma9Vdelta2 T cells. Our findings indicate that Vgamma9Vdelta2 T cells, stimulated with nanomolar concentrations of HMB-PP, strongly promote T helper type 1 (Th1) responses through their ability to induce DC maturation and IL-12 secretion. This adjuvant activity may prove useful in DC-based cancer therapies.

Fig. 1

Phenotypic properties of HMB-PP-expanded Vγ9Vδ2 T cells. Lines of Vγ9Vδ2 T cells were generated by stimulating PBMC or enriched γδ T cell fractions with 10 nM HMB-PP and culturing them in the presence of 50 U/ml IL-2. Cells were harvested on days 14–28 and analysed. a Representative flow cytometry dot plot of PBMC before (left) and after stimulation for 14 days. b Expression of CD4 and CD8 by gated Vγ9Vδ2 T cells in fresh PBMC and after expansion as above (n = 12 or 13). c Expression of activation markers CD56, CD25 and CD69 by fresh and expanded Vγ9Vδ2 T cells (n = 6–13). d Expression of naïve (CD27⁺CD45RA⁺), central memory (T_CM; CD27⁺CD45RA⁻), effector memory (T_EM; CD27⁻CD45RA⁻), and terminally differentiated (T_EMRA; CD27⁻CD45RA⁺) phenotypes by Vγ9Vδ2 T cells in fresh PBMC and expanded Vγ9Vδ2 T cells (n = 5). e Expression of molecules involved in antigen presentation, HLA-DR, CD40 and CD86 by fresh and expanded Vγ9Vδ2 T cells (n = 4–13). *p < 0.05 by paired t test analysis

Fig. 2

Functional properties of HMB-PP-expanded Vγ9Vδ2 T cells. a Frequencies of fresh and expanded Vγ9Vδ2 T cells that produced IFN-γ after stimulation (or restimulation) with medium or HMB-PP in the absence or presence of IL-2 or IL-15 (n = 2). b Killing of Daudi targets by HMB-PP-expanded Vγ9Vδ2 T cells in the absence and presence of a second stimulation with HMB-PP and IL-2 (n = 2). Error bars indicate SEM

Fig. 3

HMB-PP-expanded Vγ9Vδ2 T cells induce expression of stimulatory, costimulatory and adhesion molecules by DC. iDCs were cultured for 24 h in medium or 10 μg/ml LPS in the presence of equal numbers of fresh or HMB-PP-stimulated Vγ9Vδ2 T cells or αβ T cells. After 24 h, the cells were examined for expression of CD86, CD11c, CD54, HLA-DR, CD83, CD40, and CD80 by flow cytometry. a Representative flow cytometry histograms showing marker expression by iDC (left) and LPS-matured DC (right) in the absence and presence of HMB-PP-stimulated Vγ9Vδ2 T cells (γδ/HMB-PP). b Average mean fluorescence intensities of mAb staining of the above markers by DC incubated with medium, fresh or HMB-PP-stimulated Vγ9Vδ2 T cells or αβ T cells, or LPS. Results are means of separate experiments comparing medium with fresh γδ (n = 2), γδ/HMB-PP (n = 5), αβ (n = 3), αβ/HMB-PP (n = 2) and LPS (n = 6). Error bars indicate SEM. *p < 0.05 when compared to intensity of staining on iDC using the paired t test

Fig. 4

Activated Vγ9Vδ2 T cells induce IL-12 but not IL-10 secretion by DC. iDCs were cultured for 24 h in medium, 10 μg/ml LPS or 75 μg/ml poly I:C in the presence of equal numbers of fresh or HMB-PP-stimulated Vγ9Vδ2 T cells or αβ T cells. The levels of IL-12 and IL-10 released into the supernatants were measured by ELISA. a IL-12 and IL-10 levels secreted by DC in the absence and presence of fresh or HMB-PP-stimulated Vγ9Vδ2 T cells or αβ T cells. *p < 0.05 when compared with iDC alone using paired t test. b IL-12 and IL-10 levels secreted by LPS- or poly I:C-stimulated DC in the absence and presence of HMB-PP-stimulated Vγ9Vδ2 T cells. Results are means of separate experiments comparing medium with fresh γδ (n = 2), γδ/HMB-PP (n = 7), αβ/HMB-PP (n = 3), LPS (n = 9), LPS + γδ/HMB-PP (n = 9), poly I:C (n = 7 and poly I:C + γδ/HMB-PP (n = 7). Error bars indicate SEM. *p < 0.05 when compared with LPS-stimulated DC. Note the different scales on the two IL-12 graphs with DC and LPS-stimulated DC results shown in both

Fig. 5

DCs are not required for IFN-γ production by Vγ9Vδ2 T cells but Vγ9Vδ2 T cell-derived IFN-γ polarises DC maturation towards APC that preferentially secrete IL-12. a iDC or HMB-PP-stimulated Vγ9Vδ2 T cells (γδ/HMB-PP) were incubated separately or together in the absence or presence of 10 μg/ml LPS or 75 μg/ml poly I:C. The levels of IFN-γ released into the supernatants were measured after 24 h. Results are means of 4–7 separate experiments. ns no significant effects of adding DC were seen when analysed using the paired t test. Error bars indicate SEM. b DCs were incubated for 24 h in medium alone, 5 ng/ml IFN-γ, HMB-PP-stimulated Vγ9Vδ2 T cells (γδ/HMB-PP), 10 μg/ml LPS, LPS + IFN-γ or LPS + HMB-PP-stimulated Vγ9Vδ2 T cells. The levels of IL-12 and IL-10 released into the supernatants were measured by ELISA. Results are means of four separate experiments for IL-12 and three for IL-10. Error bars indicate SEM. *p < 0.05