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Mediators of Inflammation
Volume 2010 (2010), Article ID 704941, 11 pages
http://dx.doi.org/10.1155/2010/704941
Research Article

Preferential Th1 Cytokine Profile of Phosphoantigen-Stimulated Human Vγ9Vδ2 T Cells

1Department of Immunology and Institute of Molecular Medicine, Trinity College Dublin, St. James's Hospital, Dublin 8, Ireland
2Institute of Immunology, National University of Ireland, Maynooth, Co. Kildare, Ireland
3National Children's Research Centre, Our Lady's Hospital for Sick Children, Crumlin, Dublin 12, Ireland

Received 10 August 2010; Revised 7 December 2010; Accepted 21 December 2010

Academic Editor: Y. Mandi

Copyright © 2010 Margaret R. Dunne et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Human Vγ9Vδ2 T cells recognise pyrophosphate-based antigens (phosphoantigens) and have multiple functions in innate and adaptive immunity, including a unique ability to activate other cells of the immune system. We used flow cytometry and ELISA to define the early cytokine profiles of Vγ9Vδ2 T cells stimulated in vitro with isopentenyl pyrophosphate (IPP) and (E)-4-hydroxy-3-methyl-but-2 enyl pyrophosphate (HMB-PP) in the absence and presence of IL-2 and IL-15. We show that fresh Vγ9Vδ2 T cells produce interferon-γ (IFN-γ) and tumour necrosis factor-α (TNF-α) within 4 hours of stimulation with phosphoantigen, but neither IL-10, IL-13, nor IL-17 was detectable up to 72 hours under these conditions. Cytokine production was not influenced by expression or lack, thereof, of CD4 or CD8. Addition of IL-2 or IL-15 caused expansion of IFN-γ-producing Vγ9Vδ2 T cells, but did not enhance IFN-γ secretion after 24–72 hours. Thus, phosphoantigen-stimulated Vγ9Vδ2 T cells have potential as Th1-biasing adjuvants for immunotherapy.

1. Introduction

γδ T cells constitute approximately 1–5% of circulating human T lymphocytes, and the majority (typically >80%) of these express T-cell receptors (TCRs) consisting of Vγ9 and Vδ2 chains. The Vγ2Vδ2 TCR recognises small, nonpeptide phosphate antigens (phosphoantigens) independently of major histocompatibility complex (MHC) molecules (reviewed in references [1, 2]). This γδ T-cell subset exists only in humans and higher primates, where numerous roles in immunosurveillance and protection against infection and tumours have been hypothesised, yet a specific, unique role remains to be found. In vitro studies have shown that activated Vγ9Vδ2 T cells rapidly produce an array of proinflammatory cytokines and chemokines [3], cytolytic compounds [4, 5], antiviral [6], antimicrobial [7], and tissue growth factors [8, 9]. Activating crosstalk has been described between Vγ9Vδ2 T cells and other immune cells, such as neutrophils [10], B cells [11], monocytes [12], and dendritic cells [1316]. The Vγ9Vδ2 T-cell subset has also been shown to have antigen presentation and cross-presentation capabilities comparable to those of professional antigen presenting cells [17, 18]. The basis for this apparent functional pleiotropy is unknown but is likely to be influenced by the conditions under which γδ T cells become activated, such as in the absence or presence of an ongoing inflammatory response.

The imperative to reliably define Vγ9Vδ2 T cell responses is now more urgent, since these cells and their activating antigens are currently of interest as immunotherapeutic agents, particularly for treatment of cancer. In addition to their antigen presenting [17, 18] and adjuvant [1116] activities, Vγ9Vδ2 T cells recognise and kill a range of tumour cells in vitro [5, 1921], and clinical trials in cancer patients are ongoing [2226]. Current treatment strategies aim to optimally expand and activate Vγ9Vδ2 T cells (either ex vivo or in vivo) by administering phosphoantigens along with T-cell growth factors, most commonly IL-2, or by using aminobisphosphonates which inhibit isoprenoid synthesis resulting in the accumulation of endogenous phosphoantigens [27]. Such strategies have shown evidence of improved patient survival; however, the observed plasticity of Vγ9Vδ2 T cell functions presents a double-edged sword, with the potential to treat disease balanced against the possibility of triggering undesirable immune responses, or cell exhaustion. Thus, further characterisation of activated Vγ9Vδ2 T cells is required if such cells are to be used to generate reliable immunological outcomes.

Numerous studies have demonstrated that murine γδ T-cell subsets can produce cytokines that control distinct adaptive immune responses, such as those categorised as T helper type 1 (Th1), Th2, Th17, or regulatory T (Treg) responses [2832]. Human Vγ9Vδ2 T cells can readily release the Th1 cytokines IFN-γ and TNF-α [12, 14, 3337], and subsets of these cells can be induced under certain conditions to produce Th2 (IL-4, IL-5 and IL-13), Th17 (IL-17), and Treg (IL-10) cytokines [11, 35, 38, 39]. In the present study, we have characterised the early Th cytokine profiles of primary Vγ9Vδ2 T cells taken from the peripheral blood of healthy volunteers and stimulated in vitro with phosphoantigens in the presence and absence of T-cell growth factors IL-2 or IL-15. The antigens used were isopentenyl pyrophosphate (IPP) and (E)-4-hydroxy-3-methyl-but-2 enyl pyrophosphate (HMB-PP), intermediates of the mevalonate and nonmevalonate pathways of isoprenoid synthesis, respectively [40, 41]. HMB-PP is the most potent γδ T cell stimulating antigen described to date [40] but has yet to be clinically exploited. Our results show that Vγ9Vδ2 T cells stimulated with this novel phosphoantigen under clinically relevant conditions have similar cytokine profiles to IPP-stimulated Vγ9Vδ2 T cells, with rapid production of cytokines that promote Th1 responses, but little or no cytokines associated with Th2, Th17, or Treg responses.

2. Materials and Methods

2.1. Blood Samples and Preparation

Anticoagulated venous blood samples were obtained from healthy donors. For γδ T-cell enrichments, blood from buffy coat packs from the Irish Blood Transfusion Service Board was used. Peripheral blood mononuclear cells (PBMCs) were prepared by standard density gradient centrifugation over Lymphoprep (Nycomed, Oslo, Norway).

2.2. Magnetic Bead Enrichment of αβ and γδ T Cells

γδ T cells were enriched from PBMC by positive selection using the Anti-TCR γδ Microbead Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). T cells negative for the γδTCR (hereafter denoted αβ T cells) were prepared by positive selection of CD3+ cells from the γδ TCR-negative fractions using CD3 Microbeads (Miltenyi Biotec). Purity of αβ and γδ T-cell fractions was assessed by flow cytometry.

2.3. In Vitro Stimulation of T Cells

PBMC or enriched αβ and γδ T cells were plated at 106 cells/mL in complete RPMI medium (RPMI 1640 containing Glutamax supplemented with 25 mM HEPES, 50 mg/mL streptomycin, 50 U/mL penicillin, 2 μg/mL Fungizone, and 10% heat-inactivated foetal calf serum) and stimulated for 4–72 hours with phorbol myristate acetate (PMA; 10 ng/mL; Sigma-Aldrich, Poole, UK) and ionomycin (1 μg/mL; Sigma-Aldrich) or various concentrations of the phosphoantigens IPP (0–100 μM; Sigma-Aldrich) or HMB-PP (0-1 μM; kindly donated by Hassan Jomaa and Armin Reichenberg) [42], with and without IL-2 (50 U/mL; donated by National Cancer Institute—Frederick Research Foundation Biological Resources Branch) or IL-15 (10 ng/mL; R&D Systems, Abingdon, UK).

2.4. Intracellular Analysis of Cytokine Production by Vγ9Vδ2 T Cells

Total PBMCs were stimulated for 4 hours as described in Section 2.3 in the presence of brefeldin A (10 μg/mL, Sigma-Aldrich) to promote intracellular accumulation of cytokines. Cells were harvested and stained for cell surface expression of Vγ9, CD3, CD4, and/or CD8 and intracellular expression of IFN-γ, TNF-α, IL-10, IL-13, or IL-17 using fluorochrome-conjugated monoclonal antibodies (mAb) obtained from BD Biosciences, (Oxford, UK), Immunotools (Friesoythe, Germany), eBioscience (Hatfield, UK), and R&D Systems (Abingdon, UK) [43]. Cells were analysed using a FACSCalibur flow cytometer with CellQuest software (BD Biosciences) or with a CyAN-ADP flow cytometer with Summit (Beckman Coulter) and FloJo (Ashland, OR) software. Unstimulated control samples were incubated alongside test samples and isotype-matched, nonspecific mAbs, and fluorescence-minus-one controls were used to set compensations and gates.

2.5. Cytokine Quantification by ELISA

αβ and γδ T cells were enriched from PBMC by magnetic bead selection as described in Section 2.2 and  cells per well of a 96-well plate were stimulated in vitro as described in Section 2.3. Supernatants from these cultures were harvested 24, 48, and 72 hours after stimulation and the levels of IFN-γ, IL-10, IL-13, and IL-17 were quantified by ELISA using antibody pairs purchased from R&D Systems (DuoSet ELISA Development kits).

2.6. Statistical Analyses

Statistical analysis of data was carried out using Prism GraphPad Version 5.0. Differences between groups were assessed using paired -test, and values of <.05 were considered significant.

3. Results

3.1. Vγ9Vδ2 T Cells Rapidly Produce IFN-γ, But Not IL-10, IL-13, Nor IL-17, in Response to Pyrophosphate Stimulation

Early cytokine production by Vγ9Vδ2 T cells was examined by stimulating freshly isolated PBMC for 4 hours with medium alone, PMA and ionomycin, or various concentrations of IPP or HMB-PP in the presence of brefeldin A (10 μg/mL). Cells were then washed and stained for cell surface expression of CD3 and Vγ9 or Vδ2 and intracellular expression of IFN-γ, IL-10, IL-13, or IL-17. Virtually all Vγ9+ cells expressed the Vδ2 chain and vice versa, with single-positive Vγ9+ or Vδ2+ being very rare [16]; therefore, Vγ9Vδ2 T cells were subsequently identified by a single Vγ9 mAb stain. We found that the numbers of Vγ9+ cells detectable by flow cytometry after stimulation of PBMC with PMA and ionomycin were usually lower than those detectable in unstimulated PBMC from the same donors (not shown) or after stimulation with phosphoantigen (Figure 1). This may be due to proliferation of other non-Vγ9+ cells or downregulation of TCR expression by Vγ9Vδ2 T cells, which occurs after stimulation with PMA and ionomycin but not HMB-PP [44].

fig1
Figure 1: Vγ9Vδ2 T cells rapidly produce IFN-γ, but not IL-10, IL-13, nor IL-17, in response to PMA and ionomycin or pyrophosphate stimulation. (a) Flow cytometry dot plots showing IFN-γ, IL-10, IL-13, and IL-17 expression by Vγ9+ and Vγ9 PBMC after 4-hour stimulation with PMA and ionomycin or HMB-PP, in the presence of brefeldin A. Plots are representative of experiments on PBMC from 8 donors. Numbers in the lower right hand quadrants indicate the percentages of lymphocytes that express the Vγ9 TCR. Numbers in the upper right hand quadrants indicate the percentages of Vγ9+ T cells that produce each cytokine.

Analysis of early cytokine production by fresh PBMC from 8 healthy donors showed that Vγ9 cells produced IFN-γ, but not IL-10, IL-13, nor IL-17 within 4 hours of stimulation with either IPP (data not shown), HMB-PP (Figure 1), or PMA and ionomycin (Figure 1). The percentages of Vγ9+ cells that produced IFN-γ did not increase with greater stimulation times (data not shown). Cytokine production by non-Vγ9Vδ2 cells never exceeded background levels (0.4%) in response to phosphoantigens yet was evident in response to PMA and ionomycin treatment, for all cytokines assayed (Figure 1).

3.2. IL-2 and IL-15 Augment IFN-γ Expression But Do Not Induce IL-10, IL-13, Nor IL-17 Expression by Phosphoantigen-Stimulated Vγ9Vδ2 T Cells

The frequencies of Vγ9Vδ2 T cells that produced IFN-γ in response to stimulation of PBMC with IPP or HMB-PP were enhanced by adding IL-2 or IL-15 to culture media (Figure 2(a)). Addition of these growth factors did not lead to the induction of IL-10, IL-13, nor IL-17 production by phosphoantigen-stimulated Vγ9Vδ2+ or Vγ9Vδ2 T cells (not shown). The percentages of Vγ9Vδ2 T cells producing IFN-γ increased in a dose-dependent manner in response to IPP and HMB-PP, with half-maximal stimulation occurring when 10 μM IPP or 1 nM HMB-PP were used (Figure 2(b)). Therefore, as previously reported [40, 41], HMB-PP was ~10,000-fold more potent than IPP at stimulating IFN-γ production by Vγ9Vδ2 cells. While the presence of IL-2 or IL-15 in cell stimulations led to increases in the percentages of Vγ9Vδ2 T cells that produced IFN-γ, it did not significantly lower the concentrations of phosphoantigens that were required to induce IFN-γ expression (Figure 2(b)).

fig2
Figure 2: IL-2 and IL-15 augment IFN-γ expression by phosphoantigen-stimulated Vγ9Vδ2 T cells. (a) Representative dot plots showing IFN-γ expression by Vγ9 cells 4 hours after stimulation with IPP (10 μM) or HMB-PP (10 nM), in the absence or presence of 50 U/mL IL-2 or 10 ng/mL IL-15. Plots are representative of 8 independent studies. Numbers in the plots indicate the percentages of Vγ9 (left) and Vγ9+ (right) T cells that produce each cytokine. (b) Mean (± SEM) percentages ( ) of Vγ9 cells expressing IFN-γ after stimulation with various concentrations of IPP or HMB-PP, in the absence and presence of IL-2 and IL-15.
3.3. γδ T Cells Release Maximal Amounts of IFN-γ 72 Hours after Phosphoantigen Stimulation

We also measured levels of IFN-γ, IL-10, IL-13, and IL-17 that were released into culture supernatants of pyrophosphate-stimulated γδ TCR+ and γδ TCR (denoted αβ) T cells using ELISA. αβ and γδ T cells were enriched using magnetic beads, and cells of each type were stimulated with various concentrations of HMB-PP or IPP in the absence or presence of IL-2 or IL-15. After 24 hours, IFN-γ was detectable in the supernatants of Vγ9Vδ2 T cells, with half-maximal levels seen when 20 μM IPP or 1 nM HMB-PP were used (Figures 3(a) and 3(b)). In contrast, very low levels of IFN-γ were released by phosphoantigen-exposed αβ T cells. Measurement of IFN-γ levels 24, 48, and 72 hours after stimulation indicated that maximal amounts were detectable after 72 hours, with levels 5–10 times higher than observed after 24 hours (Figure 3(c)).

fig3
Figure 3: IFN-γ production in response to HMB-PP is restricted to γδ T cells and is highest 72 hours after simulation. Freshly isolated PBMCs were enriched for αβ and γδ T cells using magnetic beads, and 200,000 cells of each population were stimulated with various concentrations of HMB-PP (a and c) or IPP (b). The levels of IFN-γ in cell supernatants were quantified by ELISA after 24 hours (a and b) or 72 hours (c). Results are expressed as mean (± SEM) levels of IFN-γ released by αβ and γδ T cells from 2–8 donors. (d) Mean (± SEM) IFN-γ production by γδ T cells from 8 donors after stimulation with 10 μM IPP, 10 nM HMB-PP, 50 U/mL IL-2, and/or 10 ng/mL IL-15 as indicated. * compared to unstimulated cells (medium). ns: not significant comparing absence and presence of IL-2 and IL-15.

Figure 3(d) shows that stimulation of Vγ9Vδ2 T cells with IPP or HMB-PP for 72 hours resulted in significant IFN-γ release. We found that after culturing unstimulated Vγ9Vδ2 T cells for 72 hours in the presence of IL-2 or IL-15, low levels (means 0.9 and 1.2 ng/mL, resp.) of IFN-γ were detectable in the culture supernatants. However, IL-2 or IL-15 only slightly augmented the amounts of IFN-γ released by phosphoantigen-stimulated Vγ9Vδ2 T cells in 3 experiments. Thus, although IL-2 and IL-15 increase the frequencies of pyrophosphate-stimulated Vγ9Vδ2 T cells that express IFN-γ at an early time point (Figure 2), they do not significantly enhance the overall levels of IFN-γ released by these cells after 72 hours.

3.4. γδ T Cells Do Not Secrete IL-10, IL-13, Nor IL-17 in Response to Phosphoantigen Stimulation But Can Release IL-13 in Response to Treatment with IL-2 or IL-15

While enriched γδ T cells significantly upregulated IFN-γ in response to pyrophosphate stimulation (Figure 3), no upregulation of IL-10, IL-13, nor IL-17 was detected at any time point after stimulation (Figure 4). We observed that, in contrast to IFN-γ, even PMA and ionomycin treatment was insufficient to upregulate IL-10 nor IL-17 production by γδ T cells, even though these cytokines were released by PMA and ionomycin-treated total PBMC. Addition of IL-2 or IL-15 to unstimulated, IPP-stimulated, or HMB-PP-stimulated γδ T cells resulted in low level secretion of IL-13 (but not IL-10 nor IL-17) after 72 hours (Figure 4(b)). Therefore, while pyrophosphate stimulation did not induce IL-13 release by γδ T cells, some γδ T cells are nevertheless capable of producing this cytokine when cultured with the growth factors IL-2 or IL-15.

fig4
Figure 4: Phosphoantigen stimulation of γδ T cells causes no detectable increase in IL-10, IL-13, or IL-17 production. Freshly isolated PBMCs were enriched for γδ T cells using magnetic beads, and 200,000 cells were stimulated with 10 μM IPP, 10 nM HMB-PP, 50 U/mL IL-2, and/or 10 ng/mL IL-15 as indicated. The levels of IL-10 (a), IL-13 (b), and IL-17 (c) in cell supernatants were measured by ELISA after 72 hours. Bar charts show mean cytokine levels for all treatments for 8 donors. ns: not significant comparing absence and presence of IL-2 and IL-15. PMA/I-PBMC, cytokine levels released by PMA and ionomycin-stimulated PBMC.
3.5. Cytokine Production by Vγ9Vδ2 T Cells Is Independent of CD4 or CD8 Expression

Cytokine production by αβ T cells can vary according to whether they express CD4, CD8, or neither. We used flow cytometry to examine CD4 and CD8 expression by Vγ9Vδ2 T cells and found that >75% were negative for these coreceptors in 4 of 6 donors. However significant numbers of CD4+, CD8+, and double-negative Vγ9Vδ2 T cells were found in all subjects (Figures 5(a)–5(c)). We next investigated whether these subsets of Vγ9Vδ2 T cells can produce TNF-α or IFN-γ. Electronic gating on Vγ9Vδ2 T cells from 6 donors revealed that over 25% of CD4+ and CD8+ Vγ9Vδ2 T cells expressed TNF-α (Figures 5(d), 5(e), and 5(g)) and IFN-γ (Figure 5(h)) upon PMA and ionomycin stimulation. Electronic gating on CD4CD8 Vγ9Vδ2 T cells indicated that >35% of PMA and ionomycin-stimulated cells within this double-negative subset also produced these cytokines (Figures 5(f), 5(g), and 5(h)). Thus, cytokine production by Vγ9Vδ2 cells does not appear to correlate with CD4 or CD8 expression as for αβ T cells, with significant proportions of CD4+, CD8+, and double-negative Vγ9Vδ2 T cells capable of producing Th1 cytokines.

704941.fig.005
Figure 5: Cytokine expression by Vγ9Vδ2 cells is not influenced by CD4 or CD8 expression. PBMCs were stimulated with PMA and ionomycin and surface expression of Vγ9, Vδ2, CD4, and CD8, and intracellular expression of cytokines was analysed by flow cytometry after 4 hours. (a) Vγ9+Vδ2+ cells were gated initially and (b) CD4 and CD8 expression by gated Vγ9Vδ2 T cells was analysed. (c) Mean frequencies of CD4+, CD8+, and double-negative (DN) Vγ9Vδ2 T cells from 6 donors. (d, e) Flow cytometry dot plots showing expression of TNF-α by CD4+ and CD4 (d) and CD8+ and CD8 (e) Vδ2Vγ9 T cells stimulated with PMA and ionomycin. (f) Dot plot showing expression of TNF-α by gated DN Vγ9Vδ2 T cells. Arrows show electronic gates used to generate dot plots. (g, h) Frequencies of CD4+, CD8+, and DN Vδ2Vγ9 T cell that produced TNF-α (g) and IFN-γ (h) in response to stimulation with PMA and ionomycin. Results are means of 5 independent experiments.

4. Discussion

Vγ9Vδ2 T cells are innate lymphocytes that are thought to play key roles in immune sensing of danger and initiation and regulation of innate and adaptive immunity. They respond to a variety of signals including ligands for toll-like receptors [45, 46], stress-inducible ligands on infected and tumour cells [47], and phosphoantigens which may be produced endogenously or by microorganisms [1, 48]. They respond with a plethora of immunostimulatory functions, including cytotoxicity [5, 1921], direct activation of other cells of the immune system [1118], and indirect control of immune responses via the rapid secretion of chemokines and cytokines [3, 6, 7, 10, 11]. For these reasons, Vγ9Vδ2 T cells have attracted considerable interest as therapeutic agents for infectious and immune-mediated disease and cancer. Indeed, Vγ9Vδ2 T cells may perhaps be more rational candidates as immunotherapeutic targets than invariant natural killer T (iNKT) cells, which also recognise nonpeptide antigens via a conserved TCR and mediate antitumour immunity but are found at ~100-fold lower frequencies in human blood compared to Vγ9Vδ2 T cells [49].

Clinical trials using various Vγ9Vδ2 T cell activating agents, such as aminobisphosphonates and phosphoantigens, are ongoing for treatment of cancer [2226]. Such trials report low toxicity and improved objective clinical outcomes including stabilisation and partial or full remission of advanced-stage metastatic carcinomas of the prostate, lung, and kidney. Therapeutic activation of Vγ9Vδ2 T cells may also prove to be beneficial in numerous other disease settings, due to their potent adjuvant and effector functions in innate and adaptive immunity [1016]. However, it is conceivable that the same potent activities could lead to the activation of undesirable immune responses that could result in inflammatory or autoimmune disease; therefore, further characterisation of the mechanisms by which Vγ9Vδ2 T cells respond to different stimuli is required.

In this study, we analysed the cytokine production profiles of Vγ9Vδ2 T cells treated in vitro in a similar manner to current immunotherapeutic protocols, that is, using phosphoantigens and T-cell growth factors. We used two phosphoantigens, the prototype IPP [48] and the more potent HMB-PP, which can induce proliferation, cytotoxicity, and cytokine secretion by Vγ9Vδ2 T cells even at subnanomolar concentrations [40, 41]. These antigens were used to stimulate freshly isolated Vγ9Vδ2 T cells in vitro, in the presence and absence of IL-2 and IL-15, and the early production of cytokines that help polarise adaptive immune responses was examined.

Our results show that Vγ9Vδ2 T cells promptly produce the proinflammatory cytokines IFN-γ and TNF-α, in response to phosphoantigen stimulation, with IFN-γ-producing cells detected as early as 4 hours after stimulation. In contrast, we were unable to detect any upregulation of IL-10, IL-13, or IL-17 production in response to phosphoantigen stimulation. This Th1 cytokine profile was observed exclusively in Vγ9Vδ2 T cells when freshly isolated PBMCs were stimulated with either IPP or HMB-PP, and intracellular cytokine expression was analysed by flow cytometry. The same profile was seen when magnetic bead-enriched γδ T cells were stimulated for up to 72 hours and released cytokine was quantified by ELISA. The frequencies of IFN-γ expressing Vγ9Vδ2 T cells and amounts of cytokine released followed dose-dependent responses to each pyrophosphate, with HMB-PP being ~10,000-fold more potent than IPP, in agreement with previous reports [41, 42].

The addition of IL-2 or IL-15 to pyrophosphate stimulations led to moderate increases in the proportions of Vγ9Vδ2 T cells that produced IFN-γ within 4 hours of stimulation, but these increases only translated into a slight enhancement in IFN-γ levels, when supernatants were analysed after 72 hours. This suggests that IL-2 and IL-15 speed up the kinetics of the Vγ9Vδ2 response, without synergising with phosphoantigens in inducing IFN-γ production, perhaps by stimulating cell division and proliferation of IFN-γ-expressing Vγ9Vδ2 T cells rather than directly stimulating IFN-γ secretion. This result may also be explained by the presence of other accessory cells, such as antigen-presenting cells [50], within the PBMC used for intracellular cytokine detection, which were absent from the enriched γδ T-cell preparations used for ELISA.

Interestingly, γδ T cells incubated with IL-2 or IL-15 in the absence of phosphoantigen stimulation released moderate amounts of IFN-γ and IL-13, but not IL-10 nor IL-17. This IFN-γ production was augmented by simultaneous stimulation with HMB-PP or IPP, but the IL-13 production was not. This suggests that phosphoantigen stimulation of Vγ9Vδ2 T cells results in secretion of IFN-γ but not IL-13, while treatment with IL-2 or IL-15 can induce secretion of both cytokines. Alternatively, the IFN-γ and IL-13 released in response to IL-2 or IL-15 may emanate from non-Vγ9Vδ2 T cells present in the enriched γδ T-cell preparations, such as Vδ1+ T cells. However, our results clearly show that Vγ9Vδ2 T cells release IFN-γ and not IL-13 in response to phosphoantigen stimulation.

Following confirmation that phosphoantigen-stimulated Vγ9Vδ2 T cells produce IFN-γ and TNF-α, we next investigated whether this cytokine profile is restricted to CD4+, CD8+, or double-negative Vγ9Vδ2 T cells. We found that all 3 subtypes of Vγ9Vδ2 T cells were found in all 6 donors tested, with double negative cells predominating. Significant proportions of all 3 Vγ9Vδ2 T cell subsets produced IFN-γ and TNF-α upon stimulation, showing CD4 or CD8 expression does not impact on cytokine production by these cells.

The present findings are in agreement with previous studies that have shown that phosphoantigen-stimulated Vγ9Vδ2 T cells release cytokines that promote Th1 responses [12, 14, 3337]. We also confirm that IL-2 or IL-15 augment Th1 cytokine production by these cells [36, 38]. We show, for the first time, that the CD4+, CD8+, and double-negative Vγ9Vδ2 T cell subsets all exhibit this Th1 cytokine profile. Our data fit with previous observations from us and others that phosphoantigen-stimulated Vγ9Vδ2 T cells possess Th1-promoting adjuvant activity for dendritic cells, inducing them to mature into antigen-presenting cells that release IL-12 but not IL-10 [14, 16], although Eberl and coworkers [12] reported that Vγ9Vδ2 T cells can induce maturation of monocytes into antigen-presenting cells that stimulated Th1 and Th17 cells. Although Vγ9Vδ2 T cells appear to be Th1 inducers, other studies have shown that subsets of these cells can release other Th cell-polarising cytokines under different conditions. Wesch and co-workers [35] reported that Vγ9Vδ2 T cells stimulated with IPP in the presence of IL-12 and anti-IL-4 mAb (Th1-priming conditions) produced IFN-γ and TNF-α, but when stimulated in the presence of IL-4 and anti-12 mAb (Th2 priming) they produced IL-4. Vermijlen and co-workers [38] reported that Vγ9Vδ2 T cells stimulated with HMB-PP and IL-2 can also produce IL-5 and IL-13, but in agreement with our results, these authors also found that treatment of Vγ9Vδ2 T cells with IL-2 in the absence of phosphoantigen stimulation was sufficient to induce IL-13 production [38]. A minor subset of Vγ9Vδ2 T cells characterised by the expression of CXCR5 and CD27 has also been reported to release IL-4 and IL-10 following stimulation with a synthetic phosphoantigen [11]. IL-17 and IL-22 production by a minority (<0.1%) of Vγ9Vδ2 T cells stimulated with HMB-PP in the presence of IL-1β, TGF-β and IL-6, or IL-23 has also been reported [39]. Thus, it is clear that some Vγ9Vδ2 T cells can be induced to produce various cytokines, but whether they do so under physiological conditions is unclear. Furthermore, such cells appear to account for very small proportions of peripheral blood Vγ9Vδ2 T cells, which under the conditions used in the present study make very small contributions to the overall cytokine profile of phosphoantigen-stimulated Vγ9Vδ2 T cells.

In conclusion, we have shown here that IPP and the novel phosphoantigen HMB-PP rapidly activates a strong proinflammatory response specific to Vγ9Vδ2 T cells and thus shows promise as an immunotherapeutic agent, worthy of further evaluation. Furthermore, we confirm that Vγ9Vδ2 T cells are also responsive to lymphocyte growth factors. These responses seen with fresh, uncultured Vγ9Vδ2 T cells are similar to those seen with Vγ9Vδ2 T cells that were expanded in vitro with HMB-PP and IL-2 [16]. Future studies are required to ascertain the effects of stimulating Vγ9Vδ2 T cells through other non-TCR stimulatory receptors, such as toll-like receptors [45, 46] and NKG2D [47] and inhibitory receptors [51], and the effects of ligating various costimulatory and inhibitory receptors. Clarification of these responses may allow selective induction of particular Vγ9Vδ2 T-cell subsets or functions and thus greatly enhance the gamut of immunotherapeutic tools available.

Acknowledgments

The authors thank Hassan Jomaa, Armin Reichenberg, and Matthias Eberl for providing HMB-PP. Thanks to Conleth Feighery, John Jackson, Jacinta Kelly and Andrew Hogan, and Melissa Conroy and Shijuan Grace Zeng for helpful discussions. This work was supported by grants from the Irish Research Council for Science Engineering and Technology, the Health Research Board, and Science Foundation Ireland.

References

  1. C. T. Morita, C. Jin, G. Sarikonda, and H. Wang, “Nonpeptide antigens, presentation mechanisms, and immunological memory of human Vγ2Vδ2 T cells: discriminating friend from foe through the recognition of prenyl pyrophosphate antigens,” Immunological Reviews, vol. 215, no. 1, pp. 59–76, 2007. View at Publisher · View at Google Scholar · View at PubMed · View at Scopus
  2. B. Moser and M. Eberl, “γδ T cells: novel initiators of adaptive immunity,” Immunological Reviews, vol. 215, no. 1, pp. 89–102, 2007. View at Publisher · View at Google Scholar · View at PubMed · View at Scopus
  3. B. Cipriani, G. Borsellino, F. Poccia et al., “Activation of C-C β-chemokines in human peripheral blood γδ T cells by isopentenyl pyrophosphate and regulation by cytokines,” Blood, vol. 95, no. 1, pp. 39–47, 2000. View at Scopus
  4. F. Dieli, M. Troye-Blomberg, J. Ivanyi et al., “Granulysin-dependent killing of intracellular and extracellular Mycobacterium tuberculosis by V-γ9/Vδ2 T lymphocytes,” Journal of Infectious Diseases, vol. 184, no. 8, pp. 1082–1085, 2001. View at Publisher · View at Google Scholar · View at PubMed · View at Scopus
  5. P. Wrobel, H. Shojaei, B. Schittek et al., “Lysis of a broad range of epithelial tumour cells by human γδ T cells: involvement of NKG2D ligands and T-cell receptor- versus NKG2D-dependent recognition,” Scandinavian Journal of Immunology, vol. 66, no. 2-3, pp. 320–328, 2007. View at Publisher · View at Google Scholar · View at PubMed · View at Scopus
  6. T. Lehner, E. Mitchell, L. Bergmeier, et al., “The role of γδ T cells in generating antiviral factors and β-chemokines in protection against mucosal simian immunodeficiency virus infection,” European Journal of Immunology, vol. 30, no. 8, pp. 2245–2256, 2000.
  7. S. Dudal, C. Turriere, S. Bessoles et al., “Release of LL-37 by activated human Vγ9Vδ2 T cells: a microbicidal weapon against Brucella suis,” Journal of Immunology, vol. 177, no. 8, pp. 5533–5539, 2006. View at Scopus
  8. G. Workalemahu, M. Foerster, and C. Kroegel, “Expression and synthesis of fibroblast growth factor-9 in human γδ T-lymphocytes. Response to isopentenyl pyrophosphate and TGF-β1/IL-15,” Journal of Leukocyte Biology, vol. 75, no. 4, pp. 657–663, 2004. View at Publisher · View at Google Scholar · View at PubMed · View at Scopus
  9. G. Workalemahu, M. Foerster, C. Kroegel, and R. K. Braun, “Human γδ-T lymphocytes express and synthesize connective tissue growth factor: effect of IL-15 and TGF-β1 and comparison with αβ-T lymphocytes,” Journal of Immunology, vol. 170, no. 1, pp. 153–157, 2003. View at Scopus
  10. C. Agrati, E. Cimini, A. Sacchi et al., “Activated Vγ9Vδ2 T cells trigger granulocyte functions via MCP-2 release,” Journal of Immunology, vol. 182, no. 1, pp. 522–529, 2009. View at Scopus
  11. N. Caccamo, L. Battistini, M. Bonneville et al., “CXCR5 identifies a subset of Vγ9Vδ2 T cells which secrete IL-4 and IL-10 and help B cells for antibody production,” Journal of Immunology, vol. 177, no. 8, pp. 5290–5295, 2006. View at Scopus
  12. M. Eberl, G. W. Roberts, S. Meuter, J. D. Williams, N. Topley, and B. Moser, “A rapid crosstalk of human γδ T cells and monocytes drives the acute inflammation in bacterial infections,” PLoS Pathogens, vol. 5, no. 2, Article ID e1000308, 2009. View at Publisher · View at Google Scholar · View at PubMed · View at Scopus
  13. J. Ismaili, V. Olislagers, R. Poupot, J. J. Fournié, and M. Goldman, “Human γδ T cells induce dendritic cell maturation,” Clinical Immunology, vol. 103, no. 3, pp. 296–302, 2002. View at Publisher · View at Google Scholar · View at Scopus
  14. L. Conti, R. Casetti, M. Cardone et al., “Reciprocal activating interaction between dendritic cells and pamidronate-stimulated γδ T cells: role of CD86 and inflammatory cytokines,” Journal of Immunology, vol. 174, no. 1, pp. 252–260, 2005. View at Scopus
  15. M. C. Devilder, S. Maillet, I. Bouyge-Moreau, E. Bonnadieu, M. Bonneville, and E. Scotet, “Potentiation of antigen-stimulated Vγ9Vδ2 T cell cytokine production by immature dendritic cells (DC) and reciprocal effect on DC maturation,” Journal of Immunology, vol. 176, no. 3, pp. 1386–1393, 2006. View at Scopus
  16. M. R. Dunne, L. Madrigal-Estebas, L. M. Tobin, and D. G. Doherty, “(E)-4-hydroxy-3-methyl-but-2 enyl pyrophosphate-stimulated Vγ9Vδ2 T cells possess T helper type 1-promoting adjuvant activity for human monocyte-derived dendritic cells,” Cancer Immunology, Immunotherapy, vol. 59, no. 7, pp. 1109–1120, 2010. View at Publisher · View at Google Scholar · View at PubMed · View at Scopus
  17. M. Brandes, K. Willimann, and B. Moser, “Immunology: professional antigen-presentation function by human γδ cells,” Science, vol. 309, no. 5732, pp. 264–268, 2005. View at Publisher · View at Google Scholar · View at PubMed · View at Scopus
  18. S. Meuter, M. Eberl, and B. Moser, “Prolonged antigen survival and cytosolic export in cross-presenting human γδ T cells,” Proceedings of the National Academy of Sciences of the United States of America, vol. 107, no. 19, pp. 8730–8735, 2010. View at Publisher · View at Google Scholar · View at PubMed · View at Scopus
  19. P. Fisch, M. Malkovsky, S. Kovats et al., “Recognition by human Vγ9Vδ2 T cells of a GroEL homolog on Daudi Burkitt's lymphoma cells,” Science, vol. 250, no. 4985, pp. 1269–1273, 1990. View at Scopus
  20. E. Viey, G. Fromont, B. Escudier et al., “Phosphostim-activated γδ T cells kill autologous metastatic renal cell carcinoma,” Journal of Immunology, vol. 174, no. 3, pp. 1338–1347, 2005. View at Scopus
  21. S. R. Mattarollo, T. Kenna, M. Nieda, and A. J. Nicol, “Chemotherapy and zoledronate sensitize solid tumour cells to Vγ9Vδ2 T cell cytotoxicity,” Cancer Immunology, Immunotherapy, vol. 56, no. 8, pp. 1285–1297, 2007. View at Publisher · View at Google Scholar · View at PubMed · View at Scopus
  22. M. Wilhelm, V. Kunzmann, S. Eckstein et al., “γδ T cells for immune therapy of patients with lymphoid malignancies,” Blood, vol. 102, no. 1, pp. 200–206, 2003. View at Publisher · View at Google Scholar · View at PubMed · View at Scopus
  23. J. Bennouna, E. Bompas, E. M. Neidhardt et al., “Phase-I study of Innacell γδ, an autologous cell-therapy product highly enriched in Vγ9Vδ2 T lymphocytes, in combination with IL-2, in patients with metastatic renal cell carcinoma,” Cancer Immunology, Immunotherapy, vol. 57, no. 11, pp. 1599–1609, 2008. View at Publisher · View at Google Scholar · View at PubMed
  24. H. Kobayashi, Y. Tanaka, H. Shimmura, N. Minato, and K. Tanabe, “Complete remission of lung metastasis following adoptive immunotherapy using activated autologous γδ T-cells in a patient with renal cell carcinoma,” Anticancer Research, vol. 30, no. 2, pp. 575–579, 2010.
  25. J. Nakajima, T. Murakawa, T. Fukami et al., “A phase I study of adoptive immunotherapy for recurrent non-small-cell lung cancer patients with autologous γδ T cells,” European Journal of Cardio-Thoracic Surgery, vol. 37, no. 5, pp. 1191–1197, 2010. View at Publisher · View at Google Scholar · View at PubMed
  26. F. Dieli, D. Vermijlen, F. Fulfaro et al., “Targeting human γδ T cells with zoledronate and interleukin-2 for immunotherapy of hormone-refractory prostate cancer,” Cancer Research, vol. 67, no. 15, pp. 7450–7457, 2007. View at Publisher · View at Google Scholar · View at PubMed
  27. H. J. Gober, M. Kistowska, L. Angman, P. Jenö, L. Mori, and G. De Libero, “Human T cell receptor γδ cells recognize endogenous mevalonate metabolites in tumor cells,” Journal of Experimental Medicine, vol. 197, no. 2, pp. 163–168, 2003. View at Publisher · View at Google Scholar · View at Scopus
  28. D. A. Ferrick, M. D. Schrenzel, T. Mulvania, B. Hsieh, W. C. Ferlin, and H. Lepper, “Differential production of interferon-γ and interleukin-4 in response to Th1- and Th2-stimulating pathogens by γδ T cells in vivo,” Nature, vol. 373, no. 6511, pp. 255–257, 1995. View at Scopus
  29. L. Wen, D. F. Barber, W. Pao, F. S. Wong, M. J. Owen, and A. Hayday, “Primary γδ cell clones can be defined phenotypically and functionally as Th1/Th2 cells and illustrate the association of CD4 with Th2 differentiation,” Journal of Immunology, vol. 160, no. 4, pp. 1965–1974, 1998. View at Scopus
  30. A. Hayday and R. Tigelaar, “Immunoregulation in the tissues by γδ T cells,” Nature Reviews Immunology, vol. 3, no. 3, pp. 233–242, 2003. View at Publisher · View at Google Scholar · View at PubMed · View at Scopus
  31. B. Martin, K. Hirota, D. J. Cua, B. Stockinger, and M. Veldhoen, “Interleukin-17-producing γδ T cells selectively expand in response to pathogen products and environmental signals,” Immunity, vol. 31, no. 2, pp. 321–330, 2009. View at Publisher · View at Google Scholar · View at PubMed · View at Scopus
  32. C. E. Sutton, S. J. Lalor, C. M. Sweeney, C. F. Brereton, ED. C. Lavelle, and K. H. G. Mills, “Interleukin-1 and IL-23 induce innate IL-17 production from γδ T cells, amplifying Th17 responses and autoimmunity,” Immunity, vol. 31, no. 2, pp. 331–341, 2009. View at Publisher · View at Google Scholar · View at PubMed · View at Scopus
  33. V. E. García, P. A. Sieling, J. Gong et al., “Single-cell cytokine analysis of γδ T cell responses to nonpeptide mycobacterial antigens,” Journal of Immunology, vol. 159, no. 3, pp. 1328–1335, 1997. View at Scopus
  34. K. Tsukaguchi, B. De Lange, and W. H. Boom, “Differential regulation of IFN-γ, TNF-α, and IL-10 production by CD4+αβTCR+ T cells and Vδ2 γδ T cells in response to monocytes infected with Mycobacterium tuberculosis-H37Ra,” Cellular Immunology, vol. 194, no. 1, pp. 12–20, 1999. View at Publisher · View at Google Scholar · View at PubMed · View at Scopus
  35. D. Wesch, A. Glatzel, and D. Kabelitz, “Differentiation of resting human peripheral blood γδ T cells toward Th1- or Th2-phenotype,” Cellular Immunology, vol. 212, no. 2, pp. 110–117, 2001. View at Publisher · View at Google Scholar · View at PubMed · View at Scopus
  36. M. Eberl, R. Engel, E. Beck, and H. Jomaa, “Differentiation of human γδ T cells towards distinct memory phenotypes,” Cellular Immunology, vol. 218, no. 1-2, pp. 1–6, 2002. View at Publisher · View at Google Scholar · View at Scopus
  37. D. F. Angelini, G. Borsellino, M. Poupot et al., “FcγRIII discriminates between 2 subsets of Vγ9Vδ2 effector cells with different responses and activation pathways,” Blood, vol. 104, no. 6, pp. 1801–1807, 2004. View at Publisher · View at Google Scholar · View at PubMed · View at Scopus
  38. D. Vermijlen, P. Ellis, C. Langford et al., “Distinct cytokine-driven responses of activated blood γδ T cells: insights into unconventional T cell pleiotropy,” Journal of Immunology, vol. 178, no. 7, pp. 4304–4314, 2007. View at Scopus
  39. K. J. Ness-Schwickerath, C. Jin, and C. T. Morita, “Cytokine requirements for the differentiation and expansion of IL-17A- and IL-22-producing human Vγ9Vδ2 T cells,” Journal of Immunology, vol. 184, no. 12, pp. 7268–7280, 2010. View at Publisher · View at Google Scholar · View at PubMed · View at Scopus
  40. M. Hintz, A. Reichenberg, B. Altincicek et al., “Identification of (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate as a major activator for human γδ T cells in Escherichia coli,” FEBS Letters, vol. 509, no. 2, pp. 317–322, 2001. View at Publisher · View at Google Scholar · View at Scopus
  41. M. Eberl, M. Hintz, A. Reichenberg, A. K. Kollas, J. Wiesner, and H. Jomaa, “Microbial isoprenoid biosynthesis and human γδ T cell activation,” FEBS Letters, vol. 544, no. 1–3, pp. 4–10, 2003. View at Publisher · View at Google Scholar · View at Scopus
  42. A. Reichenberg, M. Hintz, Y. Kletschek et al., “Replacing the pyrophosphate group of HMB-PP by a diphosphonate function abrogates its potential to activate human γδ T cells but does not lead to competitive antagonism,” Bioorganic and Medicinal Chemistry Letters, vol. 13, no. 7, pp. 1257–1260, 2003. View at Publisher · View at Google Scholar · View at Scopus
  43. D. G. Doherty, S. Norris, L. Madrigal-Estebas et al., “The human liver contains multiple populations of NK cells, T cells, and CD3+CD56+ natural T cells with distinct cytotoxic activities and Th1, Th2, and Th0 cytokine secretion patterns,” Journal of Immunology, vol. 163, no. 4, pp. 2314–2321, 1999. View at Scopus
  44. D. V. Correia, F. D'Orey, B. A. Cardoso et al., “Highly active microbial phosphoantigen induces rapid yet sustained MEK/Erk- and PI-3K/Akt-mediated signal transduction in anti-tumor human γδ T-cells,” PLoS One, vol. 4, no. 5, Article ID e5657, 2009. View at Publisher · View at Google Scholar · View at PubMed · View at Scopus
  45. K. Pietschmann, S. Beetz, S. Welte et al., “Toll-like receptor expression and function in subsets of human γδ T lymphocytes,” Scandinavian Journal of Immunology, vol. 70, no. 3, pp. 245–255, 2009. View at Publisher · View at Google Scholar · View at PubMed · View at Scopus
  46. H. Shojaei, H. H. Oberg, M. Juricke et al., “Toll-like receptors 3 and 7 agonists enhance tumor cell lysis by human γδ T cells,” Cancer Research, vol. 69, no. 22, pp. 8710–8717, 2009. View at Publisher · View at Google Scholar · View at PubMed · View at Scopus
  47. S. Bauer, V. Groh, J. Wu et al., “Activation of NK cells and T cells by NKG2D, a receptor for stress-inducible MICA,” Science, vol. 285, no. 5428, pp. 727–729, 1999. View at Publisher · View at Google Scholar · View at Scopus
  48. Y. Tanaka, C. T. Morita, Y. Tanaka, E. Nieves, M. B. Brenner, and B. R. Bloom, “Natural and synthetic non-peptide antigens recognized by human γδ T cells,” Nature, vol. 375, no. 6527, pp. 155–158, 1995. View at Scopus
  49. A. Bendelac, P. B. Savage, and L. Teyton, “The biology of NKT cells,” Annual Review of Immunology, vol. 25, pp. 297–336, 2007. View at Publisher · View at Google Scholar · View at PubMed · View at Scopus
  50. C. T. Morita, E. M. Beckman, J. F. Bukowski et al., “Direct presentation of nonpeptide prenyl pyrophosphate antigens to human γδ T cells,” Immunity, vol. 3, no. 4, pp. 495–507, 1995. View at Publisher · View at Google Scholar · View at Scopus
  51. X. Lafarge, V. Pitard, S. Ravet et al., “Expression of MHC class I receptors confers functional intraclonal heterogeneity to a reactive expansion of γδ T cells,” European Journal of Immunology, vol. 35, no. 6, pp. 1896–1905, 2005. View at Publisher · View at Google Scholar · View at PubMed · View at Scopus