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Originally published online as doi:10.1189/jlb.0307185 on December 21, 2007

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(Journal of Leukocyte Biology. 2008;83:742-754.)
© 2008 by Society for Leukocyte Biology

Copulsing tumor antigen-pulsed dendritic cells with zoledronate efficiently enhance the expansion of tumor antigen-specific CD8+ T cells via V{gamma}9{gamma}{delta} T cell activation

Masashi Takahara*, Manami Miyai*, Mai Tomiyama*, Masato Mutou*, Andrew J. Nicol{dagger} and Mie Nieda*,1

* Medinet Medical Institute, Tokyo, Japan; and
{dagger} Centre for Immune and Targeted Therapy, University of Queensland, Greenslopes Private Hospital, Brisbane, Australia

1Correspondence: Medinet Medical Institute, Medinet Co., Ltd., 4-20-18 Seta, Setagaya-ku, Tokyo, 158-0095, Japan. E-mail: nieda{at}medinet-inc.co.jp

ABSTRACT

We demonstrate that V{gamma}9{gamma}{delta} T cells activated by zoledronate can link innate and acquired immunity through crosstalk with dendritic cells (DCs) in a way that can amplify activation and proliferation of tumor antigen-specific CD8+ T cells. DCs pulsed with antigen alone or antigen plus zoledronate were used to stimulate the in vitro expansion of antigen-specific CD8+ T cells. MART-1-modified peptide (A27L peptide) and apoptotic HLA-A*0201-positive, MART-1-positive JCOCB tumor cell lines were used as tumor antigen sources. The percentage of A27L-specific CD8+ T cells within the responding lymphocytes on Day 7 when immature DCs (imDCs) were cultured in the presence of A27L peptide and 0.01 µM zoledronate was significantly higher (P=0.002, n=11) than that observed when imDCs were cultured with the lymphocytes in the presence of the A27L peptide alone. This enhancing effect of zoledronate was significantly reduced when {gamma}{delta} T cells were depleted from responding lymphocytes (P=0.030, n=5), indicating that the effect is mediated mainly through V{gamma}9{gamma}{delta} T cells activated by zoledronate-pulsed imDCs. When imDCs copulsed with zoledronate and apoptotic JCOCB tumor cell lines were used, the percentage of A27L-specific CD8+ T cells was higher than that observed using imDCs with the apoptotic JCOCB lines alone, suggesting that zoledronate treatment of imDCs enhances the cross-presentation ability of DCs. These findings suggest a potentially valuable role for V{gamma}9{gamma}{delta} T cell activation for expanding antigen-specific CD8+T cells using DCs copulsed with tumor antigen and zoledronate in the design of vaccine therapies for malignancy.

Key Words: human • CTLs • adjuvants • aminobisphophonates

INTRODUCTION

Immunotherapy against tumors or infectious agents is based on the concept that a rapid or vigorous, therapeutically induced immune response will be able to effectively control tumors or infectious agents not eradicated by the natural immune response. New methods to increase the immune response and a greater understanding of all components of the antigen-specific immune response are needed for further therapeutic progress. CTLs are the most efficient cells for specifically and directly killing malignant or infected cells. Therefore, one of the goals of an effective immunotherapy against cancer or infectious diseases is the induction of a strong antigen-specific CTL response [1 , 2 ]. Dendritic cell (DC) therapy, aimed at stimulating tumor- or viral antigen-specific CTL in vivo, frequently induces a measurable immune response. However, clinical responses are seen in a minority of patients, presumably, at least in part, as a result of insufficient expansion of antigen-specific CTLs capable of eradicating tumor cells or virally infected cells. Despite major advances in our understanding of the adaptive immune system’s activity against tumors or infectious agents, many conventional vaccine delivery systems and adjuvants approved for human use are relatively poor at inducing antigen-specific CD8+ CTL responses [1 2 3 4 5 6 ].

Immune responses are generally divided into innate and acquired responses. Accumulating evidence indicates that multiple interactions commonly occur between immune effector cells, and these are crucial for the initiation as well as the outcome of these immune responses [7 ]. For successful immunotherapy, it is crucial to marshal and orchestrate a vast array of effector cells, including conventional {alpha}β T cells (CTLs), {gamma}{delta} T cells, NK cells, and invariant NKT cells (iNKT cells). DCs play a pivotal role in bridging innate and acquired immunity by facilitating crosstalk between such effector cells, directly or indirectly through cytokines derived from effector cells and DCs.

This interplay provides opportunities for immune effector cells to play an adjuvant role, enhancing antigen-specific {alpha}β T cell responses. Adjuvant effects of iNKT cell activation on antigen-specific CTLs have been demonstrated in murine models [8 ]. We have recently shown that the iNKT cells activated by {alpha}-galactosylceramide-pulsed DCs can bridge innate and acquired immunity to induce activation and proliferation of IFN-{gamma}-producing CD8+ T cells in the human system [9 ]. Also, it has been shown that NK cells interact with DCs and engage in an active crosstalk, inducing DC maturation through cytokines secreted by NK cells, thereby promoting acquired immune responses [10 , 11 ]. Characterization of {gamma}{delta} T cells provided the first evidence for a lymphocyte subset that could interface innate and acquired immunity [7 , 12 , 13 ]. However, it has not been clearly demonstrated that {gamma}{delta} T cells can also interact with DCs in a way that results in active crosstalk inducing acquired immune responses, in particular, induction of antigen-specific CTL responses.

Since the fortuitous discovery of {gamma}{delta} T cells about two decades ago, many articles have been written that address the biology of these lymphocytes in animal and human models [14 ]. In humans, most of our knowledge about the specificity and biological role of {gamma}{delta} T cells is derived from analysis of a major peripheral subset referred to as V{gamma}9V{delta}2 T cells, comprising 5–10% of all circulating T cells. There is growing evidence for cytotoxic antitumor activities of V{gamma}9V{delta}2 T cells against a large range of tumor types [15 16 17 ]. The V{gamma}9V{delta}2 T cells express and use NK cell-activating receptors, such as NKG2D, recognizing targets expressing stress-inducible NKG2D ligands MHC class I chain-related antigens A and B (MICA and MICB) and UL-16-binding proteins [18 19 20 ]. After target cell recognition, cytotoxicity is generally mediated by the perforin/granzyme pathway [21 , 22 ]. Unlike classical {alpha}β T cells, the V{gamma}9V{delta}2 T cells can interact with low molecular mass phosphate-containing, nonprocessed antigens, such as pyrophosphomonoesters [23 ] and alkyl amine [24 ]. Recently, aminobisphosphonates, such as pamidronate and zoledronate, were also shown to activate V{gamma}9V{delta}2 T cells, and the aminobisphosphonate-activated V{gamma}9V{delta}2 T cells were functionally characterized in vitro and in vivo [25 26 27 28 29 ]. The activated V{gamma}9V{delta}2 T cells rapidly release Th1 cytokine such as IFN-{gamma} and TNF-{alpha}, enhancing antitumor activity by inhibiting tumor growth and activating components of the adaptive immune system [30 , 31 ]. In contrast to pyrophosphomonoesters, the V{gamma}9V{delta}2 T cell activation by aminobisphophonates is highly dependent on the presence of APCs including DCs [32 33 34 ]. Aminobisphosphonates appear to target the mevalonate pathway of the APCs and induce the accumulation of phosphorylated metabolites, such as isopentenyl pyrophosphate, which are recognized by the V{gamma}9V{delta}2 T cells. As described above, it is well known that aminobisphosphonate-activated V{gamma}9V{delta}2 T cells have potent antitumor cytotoxicity and that DCs act as cellular bridges between innate and acquired immunity, interacting with NK cells or iNKT cells. However, little is known about whether aminobisphosphonate-activated V{gamma}9V{delta}2 T cell effects on DCs could modulate immune responses mediated by conventional {alpha}β T cells, in particular, by tumor antigen-specific CD8+ CTL [34 , 35 ].

The aim of this study was to investigate whether V{gamma}9V{delta}2 {gamma}{delta} T cells activated by zoledronate-pulsed DCs can link innate and acquired immunity in a way that can amplify activation and proliferation of tumor antigen-specific CD8+ T cells. We describe a role for zoledronate in efficiently enhancing DC-induced expansion of antigen-specific CD8+ CTLs and provide evidence for a role of {gamma}{delta} T cells in enhancing acquired tumor antigen-specific immunity. We highlight the implications of the cellular crosstalk between DCs and {gamma}{delta} T cells on the design of immune-based therapies for control of cancer and infection.

MATERIALS AND METHODS

Antibodies and reagents
The cell surface phenotype of {gamma}{delta} T cells, {alpha}β T cells, and DCs was determined by single or two-color flow cytometry using the Epics XL MCL (Beckman Coulter, Erembodegem, Belgium), and data were analyzed using ADC software (Beckman Coulter). The following mAb were used for flow cytometry: anti-CD14-PE, anti-HLA-DR-PE, anti-CD83-PE, anti-CD40-PE, anti-TCRV{gamma}9-FITC, anti-TCRV{gamma}9-PE, anti-TCRV{delta}2-FITC, anti-TCR{alpha}β-PE, anti-CD3-PC5, anti-CD40 ligand (CD40L)-PE, anti-CD54-PE, anti-CD80-PE, and anti-CD62L-PE from Beckman Coulter; anti-CD8-FITC, anti-HLA-ABC-PE, and anti-CD86-PE from BD Biosciences (San Jose, CA, USA); and anti-CCR7 from R&D Systems (Minneapolis, MN, USA). The purified mAb specific for IFN-{gamma} mAb was obtained from BD Biosciences. Isotype-matched controls were also obtained from Beckman Coulter and BD Biosciences. Zoledronate was purchased from Novartis Pharmaceuticals (Basel, Swiss). HLA-A*0201-restricted, modified MART-1 (A27L) 10mer synthetic peptides (ELAGIGILTV) and A27L tetramer were obtained from Operon (Tokyo, Japan) and MBL (Nagoya, Japan), respectively. HLA-A*0201-positive, MART-1-positive tumor cell line (JCOCB) was kindly provided as a gift by Dr. Chris Schmidt at the Queensland Institute of Medical Research (Brisbane, Australia). This cell line was originally established from fresh surgical specimens.

Generation of DCs and cell culture
Peripheral blood (PB) samples were collected from healthy donors possessing HLA-A*0201. The study was approved by the Ethics Committee of the Seta Clinic (Tokyo, Japan). All subjects provided written, informed consent. PBMCs were isolated by density gradient centrifugation with Lymphoprep (Nycomed, Oslo, Norway). For cell culture in this study, AIM-V medium (Invitrogen, Tokyo, Japan), supplemented with 10% heat-inactivated human AB serum, was used. CD14-positive monocytes were purified from PBMCs by positive selection with anti-CD14 mAb using MACS (Miltenyi Biotec, Bergich Gladbach, Germany). Monocytes were cultured with 500 U/mL recombinant IL-4 (Osteogenetics GmbH, Würzburg, Germany) and 500 U/mL recombinant GM-CSF (Immunex, Richmond, CA, USA) for ~5 days to obtain what are referred to in this manuscript as immature DCs (imDCs). Mature DCs (mDCs) were generated from this population by adding a proinflammatory cytokine cocktail containing IL-1β (10 ng/mL, Chemicon International, El Segundo, CA, USA), TNF-{alpha} (10 ng/mL, BD Pharmingen, Franklin Lakes, NJ, USA), IL-6 (10 ng/mL, R&D Systems), and PGE2 (1 µg/mL, Sigma, Tokyo, Japan) for a further 48 h. The cells defined as imDCs in this study were CD14–, HLA-DR+, HLA-ABC+, CD80+, CD86+, CD40+, CD54+, CD83 (weak), and CCR7 (very weak). mDCs were CD14–, HLA-ABC++, HLA-DR++, CD80+++, CD86+++, CD40++, CD54++, CD83++, and CCR7++. CD14-negative populations following MACS were used as responding lymphocytes.

Initial assessment of V{gamma}9{gamma}{delta} T cell proliferation by zoledronate in the presence of imDCs or mDCs
PB lymphocytes (CD14-negative fraction) from healthy donors were cultured with imDCs or mDCs in the presence of zoledronate at different concentrations (0.01 µM, 0.1 µM, and 1 µM) in the presence of IL-2 (50 U/mL, Chiron Benelux B.V., The Netherlands). These concentrations of zoledronate were chosen from our previous unpublished observations, showing that the optimal concentration of zoledronate to expand V{gamma}9{gamma}{delta} T cells, when PBMCs were cultured for 7 days in the presence of zoledronate, was between 0.01 µM and 1 µM. The percentage of V{gamma}9{gamma}{delta} T cells in expanding lymphocytes was assessed on Day 7 using flow cytometry. The percentage of V{gamma}9{gamma}{delta} T cells in the PB lymphocytes varied between 0.85% and 13%, depending on individuals in this experiment. It has been reported that the V{gamma}9 chain is in most cases paired to the V{delta}2 [36 ]. We have confirmed that the phenotype of almost all (90–98%) zoledronate-activated V{gamma}9{gamma}{delta} T cells in PB lymphocytes was V{delta}2-positive in our experiments (data not shown), and therefore, anti-TCRV{gamma}9 mAb was used to detect the phenotype of {gamma}{delta} T cells throughout this study.

Assessment of adjuvant effects of zoledronate on inducing and expanding tumor antigen-specific CD8+ T cells
DCs were cultured with autologous lymphocytes in the presence of A27L peptide (2 µg/mL), with or without zoledronate at various concentrations. IL-2 (50 U/mL) was added to the cultures every 2–3 days. The final concentrations of zoledronate evaluated were 0.01 µM, 0.1 µM, and 1 µM as zoledronic acid. Briefly, 5 x 104 imDCs or mDCs were cultured in the presence or absence of zoledronate, and subsequently, A27L peptide was added. Following overnight culture, 1 x 106 autologous lymphocytes were added to the DC culture. The percentage of A27L-specific CD8+ T cells in the total lymphocytes was assessed on Days 7 and 14 using A27L/HLA-A*0201 tetramer. The total cell numbers of A27L-specific CD8+ T cells were calculated using the total cell numbers of the expanding lymphocytes on Days 7 and 14. In some experiments, the percentage of V{gamma}9{gamma}{delta} T cells as a fraction of total lymphocytes was assessed using FITC-conjugated anti-TCRV{gamma}9 mAb. The expression of CD40L on V{gamma}9{gamma}{delta} T cells was assessed on Days 0, 3, 7, and 14 following coculture of 5 x 104 imDCs with 1 x 106 autologous lymphocytes in the presence of peptide (2 µg/mL), with or without zoledronate (0.01 µM). Alternatively, imDCs were cultured overnight in the presence or absence of zoledronate (0.1 µM) with A27L peptide or apoptotic JCOCB cell lines, and then, the imDCs extensively washed with media, prior to coculturing them with autologous lymphocytes. Apoptosis of JCOCB cell lines was induced by allowing culture overgrowth, resulting in ~40% apoptotic cells, according to the percentage of Annexin V-positive cells, which were stained using an Annexin V-FITC kit (Immunotech, Marseilles, France), according to the manufacturer’s protocol using flow cytometry. During culture, IL-2 (20 U/mL) was added every 3–4 days.

To assess the phenotype of pulsed DCs following coculture with lymphocytes in the presence of zoledronate, 5 x 105 imDCs were cultured with 2.5 x 106 autologous lymphocytes in the presence of A27L peptide, with or without zoledronate (0.01 µM) for 48 h. The expression for CCR7 and CD62L molecules on DCs was assessed by flow cytometry with gating on the DCs, defined by scatter properties and lineage negativity.

Assessment of the role of {gamma}{delta} T cells in the adjuvant effects of zoledronate
To assess the role of {gamma}{delta} T cells in the adjuvant effects of zoledronate, experiments were conducted in which the numbers of {gamma}{delta} T cells and other candidate populations in the responding populations were controlled. Two different series of experiments were performed. In one series of experiments, T cells were enriched by negatively selecting for T cells using the Pan T cell isolation kit (MACS). The enriched {gamma}{delta} T cell populations were separated from the T cell-enriched populations by negative selection with anti-{alpha}β mAb using MACS. The negatively selected cells remaining after the second of these two MACS separation were predominantly (>99%) {gamma}{delta} T cells. The enriched {alpha}β T cell populations were separated from the T cell-enriched populations by negative selection using the anti-TCR{gamma}{delta} MicroBead kit (MACS). The resulting {alpha}β T cell populations, containing >99% {alpha}β T cells, were cocultured with A27L-pulsed imDCs (with or without zoledronate), and varying numbers of the enriched {gamma}{delta} T cells returned to the responder cell populations. Varying concentrations of enriched {gamma}{delta} T cells (respectively, 1x104, 2.5x104, 5x104, 10x104, and 80x104) were added to 1 x 106-purified {alpha}β T cells. This resulted in responder populations in which >99% of cells were {alpha}β T cells or {gamma}{delta} T cells in controlled, defined ratios (with {gamma}{delta} T cell percentage of ~1%, 2.5%, 5%, 10%, and 45%). The combined cell populations were cocultured with imDCs in the presence of IL-2 and A27L (2 µg/mL), with or without zoledronate (0.01 µM) for 7 days. The percentage of A27L-specific T cells was evaluated by flow cytometry. In another series of experiments, the response of lymphocyte populations, in which {gamma}{delta} T cells were depleted from responder populations by positive selection with anti-{gamma}{delta} T cell mAb using MACS, was assessed. We confirmed by flow cytometry that more than 98% of {gamma}{delta} T cells were depleted from starting lymphocytes. Stimulator imDCs were unpulsed or pulsed with A27L or A27L and zoledronate, and the pulsed imDCs were washed extensively prior to the addition of the responder populations. Cultures were performed in the presence of added IL-2 but not additional A27L or zoledronate. The percentages of A27L-specific CD8+ T cells were determined by flow cytometry after 7 days as described above.

ELISPOT assays
To evaluate the effect of zoledronate on antigen-specific T cells, imDCs were cultured with autologous lymphocytes in the presence or absence of zoledronate at 0.01 µM with A27L peptide (2 µg/mL). Following 7 days culture, the numbers of antigen-specific, IFN-{gamma}-producing cells were determined by ELISPOT after 16 h in vitro restimulation of the cultured lymphocytes using the same conditions (imDCs and A27L peptide, with or without zoledronate) as were used for the first stimulation. ELISPOT assays were conducted, in triplicate, according to the manufacturer’s instructions (Mabtech AB, Nacka Strand, Sweden). The plates were counted using the Autoimmun Diagnostika system (AID; Autoimmun Diagnostika GmbH, Strassberg, Germany), and data were analyzed using AID software.

Cytokine analysis
The levels of IFN-{gamma} in culture supernatant following coculture of imDCs with autologous lymphocytes in the presence of peptide, with or without zoledronate (0.01 µM), were assessed at 48 h and 72 h using ELISA (Immunotech), according to the manufacturer’s instructions. The assay was performed in triplicate, and the detection limit of IFN-{gamma} is 0.08 IU/mL.

CTL assay
Fluorochromasia cell-mediated cytotoxicity testing was carried out using Terascan VPC (Minerve Tech, Tokyo, Japan) as described previously [37 , 38 ] In brief, 1 x 106 of target cells (JCOCB cell lines) in 1 mL were labeled for 45 min with 10 µL calcein-AM solution (Dojindo Laboratories, Kumamoto, Japan). The target cells were washed twice and then incubated with effectors at different E:T ratios for 4 h using 96-well half-area flat plates with lid (3696, Corning Inc., Corning, NY, USA) in triplicate. Cytotoxic activity was evaluated by the release of fluorochromasia into the medium. Viability of JCOCB target cells prior to assay was more than 91%. The JCOCB target cells used in this assessment were HLA-ABC++, MICA/B+, CD86 (weak), HLA-DR-negative, and CD80-negative.

Blocking studies with mAb
To evaluate mechanisms of cytotoxic activity against JCOCB target cells, purified, blocking mAb, including anti-MHC class I (W6/32, BioLegend, San Diego, CA, USA), anti-DR (G46-6, BD PharMingen) and anti-MICA (6D4, BioLegend), were used at 10 µg/mL to block the relevant cytotoxic pathways. The antibodies were added to effector cells 30 min prior to coculture with target cells. Mouse IgG2a (BD PharMingen) was used as controls. The results were expressed as mean ± SD counts of three cultures.

Statistical analysis
P values were calculated using the paired Student’s t-test and considered significant at a P value <0.05 and highly significant at a P value <0.01.

RESULTS

imDCs have greater capacity to expand PB V{gamma}9{gamma}{delta} T cells in the presence of zoledronate than mDCs
APCs are essential for aminobisphosphonate-induced expansion of V{gamma}9V{delta}2 {gamma}{delta} T cells [32 33 34 ]. We first compared the relative capacity of imDCs and mDCs as APCs to expand {gamma}{delta} T cells using the aminobisphosphonate, zoledronate at 0.01 µM, 0.1 µM, and 1 µM. The results indicate that zoledronate-induced expansion of V{gamma}9{gamma}{delta} T cells was greater in the presence of imDCs than in the presence of mDCs at each of the three concentrations of zoledronate (Fig. 1A ). The percentage of V{gamma}9{gamma}{delta} T cells on Day 0 in the PB lymphocytes was 5%, and this did not increase following culture with imDCs or mDCs without zoledronate (Fig. 1A) . Also, V{gamma}9{gamma}{delta} T cells did not expand in the absence of APCs, such as imDCs or mDCs (data not shown), as previously shown [32 33 34 ]. Further, the expansion of V{gamma}9{gamma}{delta} T cells from four to six different donors at three different concentrations of zoledronate was significantly greater using imDC than mDC [*, P=0.001 for 0.01 µM (n=4), **, P=0.014 for 0.1 µM (n=6), and ***, P=0.009 for 1 µM (n=5; Fig. 1B )]. In view of these observations, the following experiments were mostly performed using imDCs.


Figure 1
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  		Figure 1. (A) Zoledronate-induced expansion of V{gamma}9{gamma}{delta} T cells using imDCs and mDCs. DCs (from Donor 1) were cultured with autologous-responding lymphocytes (L) in the presence of zoledronate at 0.01 µM, 0.1 µM, and 1 µM, respectively, and the percentage of V{gamma}9{gamma}{delta} T cells in the lymphocytes was assessed on Day 7 using flow cytometry. The results are representative of four to six experiments at three different concentrations with similar results. (B) Expansion of V{gamma}9{gamma}{delta} T cells was significantly greater in response to imDCs than mDCs at all three different concentrations of zoledronate [*, P=0.001 for 0.01 µM (n=4), **, P=0.014 for 0.1 µM (n=6), and ***, P=0.009 for 1 µM (n=5)]. The percentage of V{gamma}9{gamma}{delta} T cells in the responding lymphocytes (on Day 0) varied between 0.85% and 13% depending on individuals.

Zoledronate enhances imDC-induced expansion of A27L-specific CD8+T cells using A27L peptide
To examine the adjuvant effects of zoledronate on inducing and expanding tumor antigen-specific CD8+T cells, we initially assessed generation of antigen-specific CD8+ T cells using imDCs in the presence of A27L peptide (2 µg/mL), with or without zoledronate at different concentrations (0.01–10 µM) and different culture durations (7-day culture vs. 14-day culture; Fig. 2A ). Among the different concentrations evaluated, imDCs cultured with autologous lymphocytes in the presence of the A27L peptide with 0.01 µM zoledronate for 14 days resulted in the highest total numbers of A27L-specific CD8+ T cells (A27L+ cells) and the highest percentage of A27L-specific CD8+ T cells as a fraction of total lymphocytes (Fig. 2A) . The numbers and the percentage of A27L-specific CD8+ T cells within the responding lymphocytes when imDCs were cultured in the presence of A27L peptide and 0.01 µM zoledronate were ~90- and 100-fold higher, respectively, than that observed when imDCs were cultured with the lymphocytes in the presence of A27L peptide alone following 14-day culture (Fig. 2A) . Results from 11 independent experiments using PBMCs from seven different donors showed the percentage of A27L-specific CD8+ T cells stimulated by imDCs in the presence of A27L peptide and 0.01 µM zoledronate on Day 7 was significantly higher (P=0.002, n=11) than that produced by imDCs in the presence of A27L peptide alone (Fig. 2B) . In multiple independent experiments using PBMCs collected on different days from the same donor, the results were similar (data not shown). Considering the results shown in Figures 1A and 2 , most of the following experiments were performed using imDCs with 0.01 µM zoledronate. In some cases, we also evaluated zoledronate at 0.1 µM, as this concentration also had adjuvant effects (Fig. 2A) . Using higher concentration of zoledroante with 10 µM in the presence of A27L peptide, the percentage of V{gamma}9{gamma}{delta} T cells in the expanding PB lymphocytes was much higher than that with 0.01 µM zoledronate and A27L peptide; however, the percentage of A27L-specific CD8+ T cells in the expanding PB lymphocytes was much less than that with 0.01 µM zoledronate and A27L peptide (data not shown).


Figure 2
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  		Figure 2. Zoledronate enhances imDC-induced expansion of A27L-specific CD8+ T cells using A27L peptide. imDCs were cultured with autologous lymphocytes in the presence of A27L peptide (2 µg/mL), with or without zoledronate (Z) at different concentrations (0.01–10 µM). The numbers and the percentage of A27L-specific CD8+ T cells (A27L+ cells) in the proliferated lymphocytes were determined on Days 7 and 14, as described in Materials and Methods. (A) imDCs (from Donor 7), cultured with autologus lymphocytes in the presence of the A27L peptide with zoledronate (0.01 µM) for 14 days, resulted in the highest percentage of A27L-specific CD8+ T cells (A27L+ cells) and the highest numbers of A27L+ cells. A27L-specific CD8+ T cells on Day 0 in the PB lymphocytes were 0.03%. (B) The percentage of A27L-specific CD8+ T cells (A27L+ cells) stimulated by imDCs for 7 days in the presence of A27L peptide with 0.01 µM zoledronate was increased significantly in comparison with that by imDCs in the presence of A27L peptide alone. Shown are the results from 11 independent experiments using PB lymphocytes from seven different donors (P=0.002, n=11). (C) The percentage of expanding V{gamma}9{gamma}{delta} T cells in the responding lymphocytes following coculture with imDCs in the presence of A27L peptide and zoledronate increased, in parallel with the increased percentage of A27L-specific CD8+ T cells (A27L+ cells; left). IFN-{gamma}-producing cell numbers in the expanding lymphocytes increased significantly when stimulated by imDCs in the presence of A27L peptide with zoledronate in comparison with that stimulated by imDCs in the presence of A27L peptide alone (right). imDCs (Donors 5 and 6) were cultured with autologous lymphocytes in the presence or absence of zoledronate at 0.01 µM with A27L peptide (2 µg/mL). Following 7 days culture, the percentages of A27L-specific CD8+ T cells (A27L+ cells, dark, shaded bars) and V{gamma}9{gamma}{delta} T cells ({gamma}{delta} T cells, light, shaded bars) in the proliferated lymphocytes were determined using flow cytometry, and IFN-{gamma}-producing cell numbers (numbers of spots, dotted bars) per 2500 of the proliferated lymphocytes were determined using ELISPOT assay. Data are mean values of triplicate samples. (D) The percentages of A27L-specific CD8+ T cells and V{gamma}9{gamma}{delta} T cells in the proliferated lymphocytes when cultured with imDCs in the presence of A27L peptide and 0.01 µM zoledronate on Day 7 were significantly higher than those observed by imDCs in the presence of A27L peptide alone [n=13, P<0.0001 for V{gamma}9{gamma}{delta} T cells ({gamma}{delta} T cells, light, shaded bars), and P=0.0006 for A27L-specific CD8+T cells (A27L+ cells, dark, shaded bars)].

We also assessed the percentage of V{gamma}9{gamma}{delta} T cells in responding lymphocytes following coculture with imDCs in the presence of A27L peptide, with or without zoledronate. The percentage of V{gamma}9{gamma}{delta} T cells in responding lymphocytes when stimulated with imDCs in the presence of A27L peptide with 0.01 µM zoledronate for 7 days was approximately threefold higher (Donors 5 and 6) than that observed when imDCs were pulsed with A27L peptide alone (Fig. 2C , left). To evaluate the effects of zoledronate on expansion of lymphocytes, including A27L-specific CD8+ T cells and V{gamma}9{gamma}{delta} T cells, which may be associated with the cytokine production of responding lymphocytes, we assessed IFN-{gamma} production by the expanding lymphocytes using ELISPOT assays. The results showed that when imDCs were cultured in the presence of zoledronate and A27L peptide for 7 days, total IFN-{gamma}-producing cell numbers per 2500-expanding lymphocytes were two-fold higher (Donor 5) and 17-fold higher (Donor 6) than that observed when imDCs were pulsed with A27L peptide alone (Fig. 2C , right). PBMCs were cultured for 7 days with imDCs in the presence of A27L peptide and 0.01 µM zoledronate in 13 independent experiments from five donors. The results showed the percentages of A27L-specific CD8+ T cells and V{gamma}9{gamma}{delta} T cells to be significantly higher (P=0.0006 for A27L-specific CD8+ T cells and P<0.0001 for V{gamma}9{gamma}{delta} T cells) in cultures containing zoledronate than that observed in cultures containing A27Lpeptide alone (Fig. 2D) . We assessed IFN-{gamma} production by the expanding lymphocytes from two additional donors using ELISPOT assays. The results were similar to those from Donors 5 and 6 (described in the previous section), showing that when imDCs were cultured in the presence of zoledronate and A27L peptide for 7 days, total IFN-{gamma}-producing cell numbers were higher than that observed when imDCs were pulsed with A27L peptide alone (data not shown).

It has been reported that the cytotoxic activity of expanded CTLs correlates with the frequency of melanoma tetramer-binding CD8+ T cells [39 ]. However, to verify our own tetramer results, we assessed the antigen-specific cytotoxic activity of the expanding lymphocytes responding to imDCs cultured in the presence of A27L peptide with zoledronate. As it has previously been shown that A27L tetramer-positive CD8+ T cells fully cross-recognize the natural MART-1 peptide and are able to efficiently lyse MART-1-expressing tumor cell lines [40 ], we used the JCOCB cell line expressing MART-1 as target cells for this assessment. Various E:T ratios (40:1, 20:1, and 10:1) were evaluated. We also confirmed that the JCOCB cell line expressed MICA molecules, which are ligands for NKG2D on {gamma}{delta} T cells. The results show that the cytotoxic activity against the JCOCB by the lymphocytes, expanded in zoledronate containing cultures for 7 days, was greater than for the lymphocytes expanded without zoledronate (55.0±4.5% vs. 38.2±3.2%, respectively, at an E:T ratio of 40:1). The T cell populations in zoledronate-containing cultures contained higher frequencies of A27L tetramer-positive T cells (3.03%) and of V{gamma}9{gamma}{delta} T cells (5.97%) following 7 days culture than in cultures without zoledronate with 1.07% of A27L tetramer-positive cells and 4.50% of V{gamma}9{gamma}{delta} T cells (Fig. 3A ). To assess the relative contribution of antigen-specific cytotoxic CD8+ T cells and V{gamma}9{gamma}{delta} T cells in the observed cytotoxicity, blocking experiments were performed. The cytotoxic activity of expanded lymphocytes containing 3.03% of A27L tetramer-positive cells and 4.50% of V{gamma}9{gamma}{delta} T cells was assessed at an E:T ratio of 40:1 in the presence of anti-MHC class I, anti-MICA, anti-HLA-DR mAb, and mouse IgG2a antibody as controls. Inhibition of killing was 30.4 ± 0.8% and 21.2 ± 0.3% for anti-MHC class I and anti-MICA mAb, respectively. There was no inhibition in controls using anti-HLA-DR and IgG2a antibodies (Fig. 3B) . This suggests that A27L tetramer-positive CD8+ T cells contributed more to the killing of the target cells than V{gamma}9{gamma}{delta} T cells. Two individuals were evaluated with similar results being obtained.


Figure 3
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  		Figure 3. The cytotoxic activity of the expanded lymphocytes against the MART-1-positive tumor cell line, JCOCB, is higher in populations containing higher frequencies of A27L-specific CD8+ T cells (3.03%) and V{gamma}9{gamma}{delta} T cells (5.97%) following culture with imDCs in the presence of A27L peptide with zoledronate (0.01 µM; imDC/A27L+Z) in comparison with that in populations containing lower frequencies of A27L-specific CD8+ T cells (1.07%) and V{gamma}9{gamma}{delta} T cells (4.50%) following culture with imDCs in the presence of A27L peptide alone (imDC/A27L). Data are mean values of triplicate samples. Cytotoxic activity was assessed as described in Materials and Methods. The cytotoxic activity of the expanded lymphocytes containing populations of A27L-specific CD8+ T cells and V{gamma}9{gamma}{delta} T cells against JCOCB tumor cell lines was blocked by anti-MHC class I and anti-MICA mAb. The cytotoxic activity of expanded lymphocytes containing of 3.03% of A27L tetramer-positive cells and 4.50% of V{gamma}9{gamma}{delta} T cells against JCOCB tumor cell lines was assessed at an E:T ratio of 40:1 in the presence of anti-MHC class I, anti-MICA, anti-HLA DR mAb, and mouse IgG2a antibody as controls.

V{gamma}9{gamma}{delta} T cells play a crucial role in the zoledronate-induced adjuvant effects for expansion of A27L-specific CD8+ T cells
To investigate a role of {gamma}{delta} T cells in the increased expansion of A27L-specific CD8+ T cells, imDCs were cultured with autologous-responding T cells consisting of {alpha}β T cells and {gamma}{delta} T cells in different ratios for 7 days. The results show that the numbers of A27L-specific CD8+ T cells generated from three donors increased in parallel with the increased percentage of {gamma}{delta} T cells added in the responding T cells ({alpha}β T cells+{gamma}{delta} T cells) in the presence of zoledronate but not in the absence of zoledronate (Fig. 4A ). This suggests that the activation/expansion of V{gamma}9{gamma}{delta} T cells plays a crucial role of V{gamma}9{gamma}{delta} T cells in the zoledronate-induced adjuvant effects on expansion of A27L-specific CD8+ T cells. To further confirm the role of {gamma}{delta} T cells in the zoledronate-induced adjuvant effects through DCs, {gamma}{delta} T cells were depleted from "unmanipulated" responding lymphocytes. As shown in Figure 4B , the enhancing effect of zoledronate was reduced significantly when {gamma}{delta} T cells were depleted from lymphocytes when imDCs were cultured in the presence of peptide with zoledronate for 7 days (P=0.030, n=5, using PBMCs from three donors), indicating again that the adjuvant effects of zoledronate are mediated primarily through zoledronate-activated V{gamma}9{gamma}{delta} T cells. This enhancing effect of zoledronate-activated V{gamma}9{gamma}{delta} T cells was reduced significantly when anti-IFN-{gamma} mAb was added in the coculture medium (P=0.016, n=5; Fig. 4B ). These results indicate that although other cytokines may also be involved in zoledronate-induced adjuvant effects, IFN-{gamma} induced by activated V{gamma}9{gamma}{delta} T cells seems to be a critical cytokine. In this experiment, the percentage of V{gamma}9{gamma}{delta} T cells in unmanipulated-responding lymphocytes was ~4.29 ± 3.50%, and that in V{gamma}9{gamma}{delta} T cell-depleted, responding lymphocytes was 0.50 ± 0.36%.


Figure 4
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  		Figure 4. (A) The number of A27L-specific CD8+ T cells (A27L+ cells) generated increased in parallel with the increased percentage of {gamma}{delta} T cells added in the responding T cells ({alpha}β T cells and {gamma}{delta} T cells) when cultured with imDCs in the presence of A27L peptide (P) and zoledronate (Z). Final percentages of {gamma}{delta} T cells in the responding T cell were ~1%, 2.5%, 5%, 10% and 45%. imDCs, {alpha}β T cells and {gamma}{delta} T cells were prepared from Donors 07, 08 and 10, respectively. (B) The enhancing effect of zoledronate was decreased significantly when {gamma}{delta} T cells were depleted from responding lymphocytes (L-{gamma}{delta} T; P=0.030) and was reduced significantly when anti-IFN-{gamma} was added into the culture medium (P=0.016). imDCs were cultured with autologous lymphocytes at 0.01 µM and A27L peptide. In blocking experiments, anti-IFN-{gamma} mAb (10 µg/mL) or isotype-matched control (IgG1) was added in the coculture medium of imDCs with autologous lymphocytes in the presence of A27L peptide and zoledronate. Following 7 days culture, expansion fold of A27L-specific CD8+ T cells (A27L+ cells) was determined. (C) There were no increases in the percentages of A27L-specific CD8+ T cells (A27L+ cells) and V{gamma}9{gamma}{delta} T cells following culture with imDCs in the presence of A27L peptide and IFN-{gamma}. imDCs were cultured with autologous lymphocytes in the presence of IFN-{gamma} (1 and 100 U/mL) with A27L peptide. Following 7 days culture, the percentages of A27L-specific CD8+ T cells (A27L+ cells) and V{gamma}9{gamma}{delta} T cells in the proliferated lymphocytes were determined.

We hypothesized that IFN-{gamma} induced by activated {gamma}{delta} T cells may play an important role in the induction of zoledronate-induced adjuvant effects. Therefore, we tested whether the percentages of A27L-specific CD8+ T cells and V{gamma}9{gamma}{delta} T cells increased when imDCs were cultured in the presence of A27L peptide (2 µg/mL) and IFN-{gamma} (1–100 U/mL). The results show that when IFN-{gamma}, at a concentration of 1 U/mL [a similar concentration to that detected in the supernatant after culturing lymphocytes with imDCs in the presence of zoledronate and A27L peptide (see below)] or higher (100 U/mL), was added directly into the culture medium, there was no increases in the percentages of both A27L-specific CD8+ T cells (Fig. 4C) .

Induction of CD40L on V{gamma}9{gamma}{delta} T cells in responding lymphocytes following coculture with imDCs in the presence of peptide with zoledronate
Whereas CD40 is expressed constitutively on DCs, macrophages and B cells, CD40L is induced on activated T cells, including CD4 T cells, CD8 T cells, iNKT cells and NK cells [41 42 43 44 45 46 47 ]. It has been clearly shown that the linkage of innate to acquired immunity through the interaction of DCs interacting with iNKT cells to induce antigen-specific CD8+ T cells requires CD40 ligation on DCs by CD40L-expressing iNKT cells [45 ]. We hypothesized that expression of CD40L on {gamma}{delta} T cells could be induced following stimulation by imDCs copulsed with zoledronate and that this contributed to enhancement of the induction of antigen-specific CD8+ T cells. To evaluate this possibility, we assessed the expression level of CD40L on V{gamma}9{gamma}{delta} T cells before stimulation (Day 0) and at various time intervals (Days 3, 7, and 14) following coculture with imDCs in the presence of A27L peptide (2 µg/mL), with or without zoledronate (0.01 µM). As shown in Figure 5A , the expression of CD40L on V{gamma}9{gamma}{delta} T cells was dependent on the duration of stimulation, and the highest expression (39.8%) was detected on Day 7 following stimulation with imDCs in the presence of A27L peptide with zoledronate. Following further culture, CD40L expression on V{gamma}9{gamma}{delta} T cells decreased, going down to 8.5% by Day 14. In contrast, the expression of CD40L on V{gamma}9{gamma}{delta} T cells in the presence of A27L peptide alone was minimally increased on Day 7, increasing from 4.6% on Day 0 to 7.6% on Day 7. Results from four independent experiments using PBMCs from three different donors showed the expression of CD40L on V{gamma}9{gamma}{delta} T cells stimulated by imDCs in the presence of A27L peptide with zoledronate on Day 7 to be significantly higher (P=0.0003) than that stimulated by imDCs in the presence of A27L peptide alone (Fig. 5B) . These results indicate that zoledronate copulsing was important for up-regulation of CD40L on V{gamma}9{gamma}{delta} T cells and consequently for efficient induction of antigen-specific CD8 T cells via CD40-expressing DCs. Although CD40 is constitutively expressed on DCs, in one donor assessed, the expression of CD40 on DCs was slightly greater based on mean fluorescent intensity (MFI) following coculture with imDCs in the presence of A27L peptide with zoledronate than that following coculture in the presence of A27L peptide alone (63.5 for imDC/A27L peptide vs. 79.8 for imDC/A27L peptide+zoledronate). Considering the results shown in Figure 5A , the numbers of V{gamma}9{gamma}{delta} T cells in the expanding lymphocytes were assessed on Days 7 and 14 following coculture with imDCs in the presence of A27L peptide (2 µg/mL) and zoledronate (0.01 µM), in parallel with the numbers of A27L-specific CD8+ T cells. Although expansion of V{gamma}9{gamma}{delta} T cells was higher on Day 7 than that on Day 14 when imDCs were copulsed with A27L peptide and zoledronate, the expansion of A27L-specific CD8+ T cells was higher on Day 14 than that on Day 7 (Fig. 5C) . The data are mean values (±SD) from triplicate cultures and are representative of three independent experiments from different donors.


Figure 5
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  		Figure 5. (A) The expression of CD40L on V{gamma}9{gamma}{delta} T cells was dependent on the duration of stimulation with imDCs in the presence of A27L peptide with zoledronate. imDCs (from Donor 7) were cultured with autologous lymphocytes in the presence of A27L peptide, with or without zoledronate at 0.01 µM. The expression of CD40L on V{gamma}9{gamma}{delta} T cells was assessed on Days 0, 3, 7, and 14 using flow cytometry. (B) The expression of CD40L on V{gamma}9{gamma}{delta} T cells stimulated by imDCs in the presence of A27L peptide with zoledronate on Day 7 was significantly higher than that stimulated by imDCs in the presence of A27L peptide alone. Shown are the results from four independent experiments using PB lymphocytes from three donors (P=0.0003, n=4). (C) Although expansion of V{gamma}9{gamma}{delta} T cells was higher on Day 7 than that on Day 14 following coculture of autologous lymphocytes with imDCs in the presence of A27L peptide and zoledronate, the expansion of A27L-specific CD8+ T cells was higher on Day 14 than that on Day 7. The total numbers of A27L-specific CD8+ T cells (A27L+ cells) and V{gamma}9{gamma}{delta} T cells were calculated using total numbers of the proliferated lymphocytes and the percentages of both populations in the lymphocytes on Days 7 and 14. (D) The expression of CD40L on V{gamma}9{gamma}{delta} T cells after culture with imDCs in the presence of A27L peptide and IFN-{gamma} was much less than that observed after culture with imDCs in the presence of A27L peptide and zoledronate. imDCs were cultured with autologous lymphocytes in the presence of A27L peptide and IFN-{gamma} (100 or 1000 U/mL), and the expression of CD40L on V{gamma}9{gamma}{delta} T cells was assessed on Day 7.

To evaluate for a possible role of IFN-{gamma} in the observed up-regulation of CD40L, we evaluated CD40L expression on V{gamma}9{gamma}{delta} T cells after culture of autologous lymphocytes with imDCs in the presence of A27L peptide (2 µg/mL) with IFN-{gamma} at two different concentrations (100 and 1000 U/mL). The results show that the expression of CD40L on V{gamma}9{gamma}Formula T cells slightly increased on Day 7 (4.6–12.8% for 100 U/mL and 4.6–15.8% for 1000 U/mL), but this was much less than that observed after culture with imDCs in the presence of A27Lpeptide and zoledronate (4.6–39.8%; Fig. 5A and 5D )

IFN-{gamma} production in the culture supernatant following coculture of imDCs with lymphocytes in the presence of A27L peptide with zoledronate
To further investigate a potential role for reciprocal activating interactions between DCs and aminobisphosphonates-stimulated {gamma}{delta} T cells [34 ], we assessed IFN-{gamma} production in the culture supernatants following coculture of imDCs with lymphocytes in the presence of A27L peptide, with or without zoledronate for 48 h and 72 h. The results show that IFN-{gamma} production was only detected in the presence of A27L peptide and zoledronate (not with A27L peptide alone), that the production of IFN-{gamma} after culture for 48 h was higher than after 72 h, and that this IFN-{gamma} production did not detect when {gamma}{delta}T cells were depleted from the responding lymphocytes (Fig. 6A ).


Figure 6
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  		Figure 6. (A) IFN-{gamma} production was only detected in the culture supernatant following 48–72 h culture of imDCs with autologous lymphocytes in the presence of A27L peptide with zoledronate. imDCs (Donor 6) were cultured with autologous lymphocytes or {gamma}{delta} T cell-depleted lymphocytes at a 1:5 ratio in the presence of A27L peptide, with or without zoledronate for 48 h or 72 h. The level of IFN-{gamma} in the coculture media was assessed at 48 h and 72 h using ELISA. Data are mean values of triplicate samples. The results are representative of three experiments with similar results. (B) Up-regulation of CCR7 and CD62L molecules on DCs following 48 h culture of imDCs with autologous lymphocytes in the presence of A27L peptide and zoledronate. imDCs were cultured with autologous lymphocytes as described in A. The expression of CCR7 and CD62L was assessed using flow cytometry. The results refer to one representative experiment of three.

The expression of the lymph node homing receptors, L-selectin (CD62L) and CCR7, on DCs was assessed following culture for 48 h of lymphocytes with imDCs in the presence of A27L peptide and zoledronate, in which IFN-{gamma} production was detected as described above. The results showed that up-regulation of CD62L was observed in all three donors assessed, and CCR7 up-regulation was observed in two out of three donors assessed. Again, depletion of V{gamma}9{gamma}{delta} T cells from the responding lymphocytes partially abrogated up-regulation of CD62L and CCR7. The results shown in Figure 6B (referring to one representative experiment from Donor 7) showed that expression of CCR7 and CD62L molecules on DCs increased following copulsing with zoledronate (13.1–68.3% for CCR7 and 8.6–24.3% for CD62L) and fell following depletion of {gamma}{delta} T cells from responding lymphocytes stimulated with imDCs copulsed with A27L peptide and zoledronate (68.3–35.6% for CCR7 and 24.3–16.6% for CD62L; Fig. 6B ).

The zoledronate-induced adjuvant effects for expanding A27L-specific CD8+ T cells using imDCs are higher than that using mDCs
Considering the potential application of this for DC therapy in clinical settings, we performed similar experiments using imDCs, which were first cotreated with zoledronate (0.1 µM) and A27L peptide (2 µg/mL) and then washed extensively before adding to the lymphocytes. The results show that the adjuvant effect on expanding A27L-specific CD8+ T cells by Day 14 was similar to that observed when the mixed lymphocyte culture was conducted with zoledronate and A27L peptide in the coculture medium (Figs. 2A and 7A ). Parallel experiments were conducted using mDCs instead of imDCs. Figure 7A illustrates that by Day 14, there was approximately a 60-fold increase with imDCs and a two-fold increase with mDCs as the percentage of A27L-specific CD8+ T cells once zoledronate was copulsed. The zoledronate-induced adjuvant effects using imDCs were significantly higher than that using mDCs for 7-day culture (7.30±3.27-fold expansion with imDCs vs. 1.83±0.23, with mDCs following copulsing of zoledronate; P=0.012, n=5; Fig. 7B ).


Figure 7
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  		Figure 7. Enhancement of tumor antigen-specific CD8+ T cells by imDCs copulsed with A27L peptide and zoledronate was higher than that by mDCs copulsed with A27L peptide and zoledronate. (A) imDCs and mDCs (from Donor 7) were cultured separately overnight in the presence or absence of zoledronate (0.1 µM) with A27L peptide (2 µg/mL). After extensive washing to remove peptide and zoledronate, the DCs were then cocultured with autologous lymphocytes. The percentage of A27L-specific CD8+ T cells was assessed on Day 14 by flow cytometry using A27L tetramers. The results are representative of five experiments with similar results. (B) The zoledronate-induced adjuvant effects using imDCs were significantly higher than that using mDCs for 7-day culture (7.32±3.27-fold expansion with imDCs vs. 1.83±0.23, with mDCs following copulsing of zoledronate; P=0.012, n=5). DCs (imDCs or mDCs), pulsed with A27L peptide alone or A27L peptide (2 µg/mL) plus zoledronate (0.1 µM), were cultured with autologous lymphocytes and assessed the percentage of A27L-specific CD8+ T cells on Day 7 by flow cytometry using A27L tetramer.

Zoledronate enhances imDC-induced expansion of A27L-specific CD8+T cells using apoptotic HLA-A*0201-positive, MART-1-positive JCOCB tumor cell lines as the antigen sources
To evaluate adjuvant effects with alternative antigen sources, we evaluated antigen-specific immune responses when apoptotic JCOCB tumor cell lines were used as the antigen source. In these experiments, 40.3% of the JCOCB were shown to be apoptotic based on the numbers of Annexin-V-positive cells (Fig. 8A ). The results were similar to those obtained when peptides were used as the antigen source. Specifically, we observed that when imDCs copulsed with zoledronate and apoptotic JCOCB tumor cell lines were used, the percentage of A27L-specific CD8+ T cells was higher than that observed using imDCs with apoptotic JCOCB tumor cell lines alone (approximately three- to five-fold higher on 14 days of culture; Fig. 8B ). In this experiment, when lymphocytes were cultured with the apoptotic tumor cell lines alone, the percentage of A27L-specific CD8+ T cells was 0.05–0.06%. We observed that when lymphocytes were stimulated with imDCs, copulsed with apoptotic JCOCB tumor cell lines and zoledronate, the MFI of A27L tetramer-positive cells was also increased in comparison with that observed using imDCs pulsed with apoptotic JCOCB tumor cell lines alone (Fig. 8B) .


Figure 8
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  		Figure 8. Enhancement of A27L-specific CD8+ T cells by imDCs copulsed with zoledronate using apoptotic, MART-1-positive, HLA-A*0201-positive JCOCB tumor cell lines as the antigen sources. (A) The percentage of apoptotic cells (40.3% as Annexin-V-positive cells) in the JCOCB tumor cell lines used in this experiment. 7-AAD, 7-Amino-actinomycin. (B) imDCs (Donors 07 and 04) were cultured overnight in the presence or absence of zoledronate at 0.1 µM with apoptotic JCOCB tumor cell lines (Tu). The percentage of A27L-specific CD8+ T cells (A27L+ cells) and MFI (M) of those A27L-specific CD8+ T cells were assessed on Day 14 using flow cytometry. The results are representative of seven experiments with similar results.

DISCUSSION

This study demonstrates potent, adjuvant effects of zoledronate on enhancing tumor antigen-specific T cells. We also demonstrate that V{gamma}9{gamma}{delta} T cells activated by zoledronate are pivotal in the adjuvant effects, linking innate and acquired immunity in a way that amplifies activation and proliferation of tumor antigen-specific CD8+ T cells. This has a number of important implications, some of which may have a significant impact on the use of aminobisphosphonates in the setting of cancer therapy.

The key role of V{gamma}9{gamma}{delta} T cells in the zoledronate-induced adjuvant effects was confirmed by a number of observations. Most significantly, addition of V{gamma}9{gamma}{delta} T cells to a purified population of responding {alpha}β T cells (>99% purity) in the presence of zoledronate enhanced the effects of zoledronate on induction of A27L-specific CD8+ T cells in a dose-dependent manner (Fig. 4A) . This finding is strengthened by the additional observation that depletion of V{gamma}9{gamma}{delta} T cells from responding lymphocytes significantly reduced the enhancing effects of zoledronate on induction of A27L-specific CD8+ T cells in response to stimulation by imDCs copulsed with zoledronate and A27L peptide (Fig. 4B) . The observation that activation of V{gamma}9{gamma}{delta} T cells, resulting in the induction of CD40L on the V{gamma}9{gamma}{delta} T cells on Day 7, occurred prior to higher expansion of A27L-specific CD8+T cells on Day 14 (Fig. 5A 5B 5C) provides additional but circumstantial evidence that V{gamma}9{gamma}{delta} T cell activation is a key event in the adjuvant activities of zoledronate on induction of A27L-specific CD8+ T cells. CD40–CD40L-mediated activation of antigen-presenting DCs by V{gamma}9{gamma}{delta} T cells may have an important role in the observed, adjuvant effects of zoledronate, as it was previously suggested that CD40 ligation on DCs by CD40L-expressing T cells, such as iNKT cells, plays an important role in the linkage of innate to acquired immunity through DCs to induce antigen-specific CD8+ T cells [45 ]. Antigen-specific T cell proliferation was greater following stimulation by imDCs copulsed with zoledronate and A27L peptide than mDCs copulsed with zoledronate and A27L peptide (Fig. 7A and 7B) . This was initially unexpected but may be linked with the fact that zoledronate-associated expansion of V{gamma}9{gamma}{delta} T cells was greater in response to stimulation with imDC than mDC (Fig. 1A and 1B) . One possible explanation for the greater effect of zoledronate-treated imDC on activation of V{gamma}9V{delta}2 {gamma}{delta} T cells is that the mevalonate pathway metabolites, such as isopentenyl pyrophosphate, which accumulate in response to aminobisphosphonates and are recognized by the V{gamma}9V{delta}2 TCR, may accumulate to a greater extent in imDC than mDC. The apparent advantage of imDC over mDC relating to normal physiology is not immediately apparent, but it is potentially crucial for vaccine approaches. These findings potentially reopen the debate regarding optimal DC choice, regardless of whether the DCs are manipulated in vitro or in vivo with protein, DNA, or RNA-based vaccines.

Not only was the percentage of A27L-specific CD8+ T cells stimulated by imDCs treated with A27L-associated antigen and zoledronate higher than following stimulation with imDCs treated with the A27L peptide alone, but also, the A27L tetramer-positive cells had higher TCR MFI, reflecting higher expression of the surface {alpha}βTCR, which recognizes the A27L peptide/HLA-A*0201 complex (Fig. 8B) . This was observed in almost all experiments performed, regardless of whether the antigen source was the A27L peptide or apoptotic MART-1-positive JCOCB tumor cell lines.

In the present study, when imDCs were cultured in the presence of zoledronate and A27L peptide, the IFN-{gamma}-producing cell number was higher than that observed when imDCs were cultured in the presence of A27L peptide alone (Fig. 2C) . As imDCs pulsed with zoledronate, and A27L peptide induced proliferation of A27L-specific CD8+ T cells as well as V{gamma}9{gamma}{delta} T cells (Fig. 2C and 2D) , it is not easy to determine which of the two populations is the main producer of IFN-{gamma}. We postulate that both populations contribute to the production of IFN-{gamma} and that A27L-specific CD8+ T cells produce IFN-{gamma} in cultures containing zoledronate partly as a result of DC maturation induced by IFN-{gamma}, produced by V{gamma}9{gamma}{delta} T cells, activated early in the culture period (Fig. 6) and partly as a result of DC maturation by direct effects of signaling through CD40L from V{gamma}9{gamma}{delta} T cells (Fig. 5) .

The enhancing effect of zoledronate was removed almost completely when anti-IFN-{gamma} mAb was added to the coculture medium (Fig. 4B) . IFN-{gamma} in the coculture supernatant was detected when unmanipulated lymphocytes were cultured with imDCs in the presence of A27L peptide and zoledronate but not detected when V{gamma}9{gamma}{delta} T cells were depleted from the lymphocytes (Fig. 6A) . Taken together, these results indicate that IFN-{gamma} is likely to be a key, soluble factor in the adjuvant effects of {gamma}{delta} T cells. Our observation that IFN-{gamma} production was higher at 48 h than at 72 h is consistent with previous studies using cytokine levels, showing rapid induction of IFN-{gamma} and TNF-{alpha} in V{delta}2{gamma}{delta} T cells (within 48 h) in response to DC stimulation [34 ]. Induction of IFN-{gamma} in {gamma}{delta} T cells by zoledronate may depend on cell contact with imDC, as previously reported using pamidronate (a molecule that is highly analogous to zoledronate) [34 ]. As our study suggested that IFN-{gamma} is a key, soluble factor for induction of a zoledronate-induced adjuvant effect, we evaluated the effect of directly adding IFN-{gamma} to the culture medium of imDCs with lymphocytes in the presence of peptide alone. The results showed that the percentages of A27L-specific CD8+ T cells and V{gamma}9{gamma}{delta} T cells did not increase and that the expression of CD40L on V{gamma}9{gamma}{delta} T cells was low in comparison with imDCs cultured in the presence of A27L peptide and zoledronate (Fig. 5A and 5B) . This suggests that the linkage of innate to acquired immunity through the interaction of DCs with V{gamma}9{gamma}{delta} T cells to efficiently induce antigen-specific CD8+ T cells requires CD40 ligation on DCs by CD40L-expressing V{gamma}9{gamma}{delta} T cells.

Our results, showing that aminobisphosphonates not only enhance V{gamma}9{gamma}{delta} T cell antitumor activity but also tumor antigen-specific T cells (Fig. 3) , provide further evidence for the potential role of the immune system in the documented clinical antitumor activity of these agents. It is conceivable that in the clinical setting, administration of aminobisphosphonates enhances specific antitumor immunity in the form of peptide antigen-specific T cells. imDCs taking up and processing tumor antigens and migrating to lymph nodes will be exposed to the administered aminobisphosphonates, providing an opportunity for costimulation of antigen-specific T cells by V{gamma}9{gamma}{delta} T cells. This hypothesis could be tested in appropriate tumor models. Even if this hypothesis of in vivo immune effects in response to aminobisphosphonates is incorrect, our results clearly demonstrate the potential for the manipulation of V{gamma}9{gamma}{delta} T cells to have a significant role in the development of tumor vaccine technology. It is likely that the physical size of the immune response is crucial to successful antitumor therapy. Tumor killing by direct cell–cell contact (as is presumed to be at least partly responsible for antitumor activities of T cells) dictates that there must be a favorable balance between immune effector cells and tumor targets for there to be a therapeutic benefit. Therefore, any strategy that increases the number of antitumor T cells above that obtainable with current vaccine strategies could be of potential clinical benefit.

The adjuvant effects by zoledronate-activated V{gamma}9{gamma}{delta} T cells on antigen-specific CD8+ T cells were similar when stimulator DCs were washed to remove extracellular zoledronate and tumor antigens, as when pulsed DCs and lymphocytes were cocultured in the presence of additional zoledronate and tumor antigens. These observations support the possibility that our in vitro observations could translate into the clinical situation if zoledronate-pulsed DCs are administered therapeutically. Our study demonstrated advantages, in vitro, of imDCs over mDCs with respect to stimulation of {gamma}{delta} T cells and the consequent adjuvant effects on T cells. Notably, even when DCs were maturated using a maturation cytokine cocktail containing IL-1β, IL-6, TNF-{alpha}, and PGE2 to produce one of the most potent DC types, zoledronate-induced adjuvant effects were still observed (Fig. 7A) . Although the expression of CCR7 and CD62L was observed following coculture of imDCs copulsed with peptide and zoledronate in some donors (Fig. 6B) , further investigation is warranted prior to selection of optimal DC for clinical studies of DCs pulsed with aminobisphosphonate and peptide, in terms of DC trafficking across endothelial cells and to lymph nodes [48 , 49 ]

Importantly, our results that zoledronate copulsing of imDCs increased A27L-specific CD8+ T cells when apoptotic tumor cell lines expressing MART-1 antigen were used as the antigen source confirm that the adjuvant effect of zoledronate can enhance responses to cross-presented tumor antigens. It has been previously shown that maximal antigen-specific CD8+ T cell activation by cross-presentation requires CD40L engagement [50 ]. It is assumed that zoledronate treatment of imDCs enhances cross-presentation by DCs, resulting from the interaction between activated {gamma}{delta} T cells expressing CD40L and DCs constitutively expressing CD40. Interestingly, the expression of CD40L on V{gamma}9{gamma}{delta} T cells when culturing with imDCs in the presence of IFN-{gamma} (1000 U/mL) was lower in comparison with imDCs cultured in the presence of zoledronate (Fig. 5A and 5D) , implying that imDCs treated with IFN-{gamma} may have less cross-presentation ability than imDCs treated with zoledronate. Further research is warranted to investigate precise mechanisms for enhancement of zoledronate-induced cross-presentation. Theoretically, zoledronate could similarly enhance the in vivo induction of tumor antigen-specific CD8+ CTLs in response to cross-presentation of antigens from apoptotic tumor cells resident in the body. It is interesting to speculate whether this may contribute to some of the observed antitumor activities of bisphosphonate therapy.

It has been shown recently that like DCs, {gamma}{delta} T cells present antigens to {alpha}β T cells [51 ]. It is also known that {gamma}{delta} T cells are found as tumor-infiltrating lymphocytes [52 ]. It is conceivable that {gamma}{delta} T cells, activated in vivo, for example, by administered zoledronate-pulsed imDCs, might play a role as APCs [51 , 53 ] but with the additional capacity to migrate into tumors (because of the migratory potential of T cells), where they can up-take apoptotic bodies from tumor cells and subsequently induce tumor antigen-specific CD8+ T cells in vivo. Furthermore, we speculate that the cytotoxic effects of activated {gamma}{delta} T cells within the tumors may contribute to the generation of apoptotic tumor cells as antigen sources. Any or all of these possibilities could explain some of the observed antitumor activities of aminobisphosphonates in improving outcomes in patients with malignancy.

The observed, favorable in vitro expansion of antigen-specific T cells in response to zoledronate-pulsed DCs occurred in the presence of added IL-2, indicating that additional, exogenous IL-2 may also be required to maximize the immunotherapeutic benefit of DCs copulsed with antigens and zoledronate in the clinical setting. There is extensive data indicating the feasibility of this, and interestingly, a clinical study involving coadministration of IL-2 and zoledronate demonstrated clinical responses to this combination in a patient with lymphoma [54 ].

Of particular note is that expansion of {gamma}{delta} T cells and tumor antigen-specific CD8+ T cells in combination could provide potential clinical advantages by targeting MHC class I-negative, but MICA/B-positive, and MHC-class I-positive tumor targets.

In summary, copulsing tumor antigen-pulsed imDCs with zoledronate leads first to the activation of V{gamma}9{gamma}Formula T cells, resulting in the induction of CD40L on V{gamma}9{gamma}Formula T cells, then triggering of secretion of cytokines such as IFN-{gamma}, and ultimately, to enhanced expansion of cytotoxic antitumor, antigen-specific CTL. We postulate that the induction of CD40L on V{gamma}9{gamma}Formula T cells activated by imDCs, copulsed with zoledronate and the cytokines, which the V{gamma}9{gamma}Formula T cells release, acts on imDCs, stimulating their functional maturation, including up-regulation of CCR7 and CD62L, resulting in the activation and proliferation of antigen-specific CD8+ T cells. This is the first demonstration of adjuvant effects of aminobisphosphonate-activated {gamma}Formula T cells on expansion of antigen-specific CD8+T cells. In doing so, we highlight what may be a physiologically and therapeutically important bridge between innate and acquired immunity. Our findings are anticipated to facilitate the design of more effective immune therapy strategies for patients with malignancy and may provide important insights into immune responses to infectious agents.

ACKNOWLEDGEMENTS

We thank Drs. Kiyoshi Yokokawa and Ryuji Maekawa for critical reading of this manuscript and valuable discussions.

Received March 23, 2007; revised November 15, 2007; accepted November 21, 2007.

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