Interplay of T-cell receptor and interleukin-2 signalling in Vγ2Vδ2 T-cell cytotoxicity

Abstract

Human peripheral blood Vγ2Vδ2 T cells are important for host defence and tumour immunity. Their unusual T-cell receptor (TCR) recognizes small molecule phosphoantigens; stimulated cells produce inflammatory cytokines and are potently cytotoxic for a variety of tumours. However, molecular mechanisms linking phosphoantigen stimulation and cytotoxicity are incompletely understood. We know that isopentenyl pyrophosphate (IPP) activates mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/Erk) and phosphoinositide 3-kinase (PI-3K)/Akt pathways; specific inhibition of Erk or Akt significantly impairs the functional response to IPP. We now show that interleukin-2 also activates MEK/Erk and PI-3K/Akt pathways but on its own, fails to induce cytokine expression or cytotoxicity. Hence, MEK/Erk and PI-3K/Akt activation are necessary but not sufficient to induce effector responses in Vγ2Vδ2 T cells and a TCR-dependent signal is still required for tumour cell killing. Cyclosporin A, an inhibitor of calcineurin, blocked calcium-dependent nuclear translocation of nuclear factor of activated T cell (NFAT) and significantly reduced IPP-induced cytokine production, degranulation and cytotoxicity. The IPP-induced calcium mobilization and NFAT translocation were necessary to activate Vγ2Vδ2 effector functions; interleukin-2, acting on the MEK/Erk pathway, regulated the strength of these responses. The TCR has a specific role in Vγ2Vδ2 T-cell killing of tumour cells, which is distinct from its role in triggering cellular proliferation in response to phosphoantigens.

Figure 1

Interleukin-2 (IL-2) promoted Erk and Akt phosphorylation in Vδ2 T cells. (a) Freshly isolated peripheral blood mononuclear cells (PBMC) contained 1–10% of Vγ2Vδ2 T cells (left panel), we generated a Vδ2 T-cell line by in vitro expansion with isopentenyl pyrophosphate (IPP) plus IL-2 and the percentage of Vγ2Vδ2 T cells was > 90% (right panel). (b) Vγ2Vδ2 T-cell lines were rested after washing twice and culturing in fresh medium for 24 hr before being treated with IPP (15 μm) or IL-2 (100 U/ml) for 10–60 min. After stimulation, cells were collected for western blotting assays with antibodies specific for signal transduction molecules. Data are representative of at least three independent experiments with PBMC from unrelated donors.

Figure 2

Phosphoantigen elicits cytokine, degranulation and cytotoxicity responses that are amplified by interleukin-2 (IL-2). (a,b) Peripheral blood mononuclear cells (PBMC) or Vγ2Vδ2 T-cell lines were treated with IL-2, isopentenyl pyrophosphate (IPP) or both for 4 hr. Interferon-γ (IFN-γ) or tumour necrosis factor-α (TNF-α) production (a) and CD107a expression (b) were detected by flow cytometry. Cells were gated on Vδ2⁺ cells. (c) Resting Vγ2Vδ2 T-cell lines were pretreated with IL-2 at various concentrations for 1 hr. The cytotoxicity of Vγ2Vδ2 T cells against Daudi (left panel) or TU167 (right panel) was evaluated at several effector to target (E : T) ratios with triplicate wells for each condition. At E : T = 10 : 1 there was a significant effect of increasing IL-2 on target cell lysis; *P < 0·05; **P < 0·01; ***P < 0·001. Data are representative of three independent experiments with PBMC from unrelated donors.

Figure 3

Only phosphoantigen induced calcium-dependent nuclear translocation of nuclear factor of activated T cells (NFAT). Resting Vδ2 T-cell lines were treated with interleukin-2 (IL-2; 100 U/ml) or isopentenyl pyrophosphate (IPP; 15 μm) for 15 min. NFAT1, NFAT4, phosphorylated signal transducer and activator of transcription 5 (STAT5) and β-actin were measured by Western blotting in cytoplasmic or nuclear extracts. Data are representative of two independent experiments with peripheral blood mononuclear cells from unrelated donors.

Figure 4

Cyclosporin A (CsA) blocks T-cell receptor (TCR) -dependent functions in Vδ2 T cells. (a,b) Resting Vγ2Vδ2 T-cell lines were pretreated with CsA for 1 hr, then stimulated with isopentenyl pyrophosphate (IPP; 15 μm) or IPP plus interleukin-2 (IL-2; 100 U/ml) for 4 hr. Interferon-γ (IFN-γ) or tumour necrosis factor-α (TNF-α) production (a) and CD107a expression (b) were detected by flow cytometry. (c) Resting Vγ2Vδ2 T-cell lines were pretreated with CsA (100 nm) or CsA + IL-2 (100 U/ml) for 1 hr. The cytotoxicity of Vγ2Vδ2 T cells against Daudi (left panel) or TU167 (right panel) targets were evaluated at several effector to target (E : T) ratios with triplicate wells for each condition. The statistical significance of specific lysis compared with control at E : T = 10 : 1 was analysed. ***P < 0·001. Data are representative of three independent experiments with peripheral blood mononuclear cells from unrelated donors.

Figure 5

Effects of ionomycin on functional responses of Vδ2 T cells. (a,b) Resting Vγ2Vδ2 T-cell lines were stimulated with ionomycin (1 μm), interleukin-2 (IL-2; 100 U/ml), isopentenyl pyrophosphate (IPP) or combinations as indicated, for 4 hr. Interferon-γ (IFN-γ) or tumour necrosis factor-α (TNF-α) production (a) and CD107a expression (b) were detected by flow cytometry. Data are representative of three independent experiments with peripheral blood mononuclear cells from unrelated donors.

Figure 6

Nuclear factor of activated T cells (NFAT) was involved in Vδ2 T-cell cytokine production and degranulation. (a,b) Resting Vγ2Vδ2 T-cell lines were pretreated with NFAT inhibitor or vehicle control (DMSO) for 1 hr, then stimulated with isopentenyl pyrophosphate (IPP; 15 μm) for 4 hr. Interferon-γ (IFN-γ) or tumour necrosis factor-α (TNF-α) production (a) and CD107a expression (b) were detected by flow cytometry. Data are representative of three independent experiments with peripheral blood mononuclear cells from unrelated donors.