The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

The Journal of Immunology, 2007, 179, 6468 -6478
Copyright © 2007 by The American Association of Immunologists, Inc.
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Holderness, J.
Right arrow Articles by Jutila, M. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Holderness, J.
Right arrow Articles by Jutila, M. A.
Right arrowPubmed/NCBI databases
*Substance via MeSH

Select Plant Tannins Induce IL-2R{alpha} Up-Regulation and Augment Cell Division in {gamma}{delta} T Cells1

Jeff Holderness*, Larissa Jackiw*, Emily Kimmel*, Hannah Kerns*, Miranda Radke*, Jodi F. Hedges*, Charles Petrie{dagger}, Patrick McCurley{dagger}, Pati M. Glee{dagger}, Aiyappa Palecanda{dagger} and Mark A. Jutila2,*

* Veterinary Molecular Biology, Montana State University, and {dagger} LigoCyte Pharmaceuticals, Inc., Bozeman, MT 59718


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
{gamma}{delta} T cells are innate immune cells that participate in host responses against many pathogens and cancers. Recently, phosphoantigen-based drugs, capable of expanding {gamma}{delta} T cells in vivo, entered clinical trials with the goal of enhancing innate immune system functions. Potential shortcomings of these drugs include the induction of nonresponsiveness upon repeated use and the expansion of only the V{delta}2 subset of human {gamma}{delta} T cells. V{delta}1 T cells, the major tissue subset, are unaffected by phosphoantigen agonists. Using FACS-based assays, we screened primary bovine cells for novel {gamma}{delta} T cell agonists with activities not encompassed by the current treatments in an effort to realize the full therapeutic potential of {gamma}{delta} T cells. We identified {gamma}{delta} T cell agonists derived from the condensed tannin fractions of Uncaria tomentosa (Cat’s Claw) and Malus domestica (apple). Based on superior potency, the apple extract was selected for detailed analyses on human cells. The apple extract was a potent agonist for both human V{delta}1 and V{delta}2 T cells and NK cells. Additionally, the extract greatly enhanced phosphoantigen-induced {gamma}{delta} T cell expansion. Our analyses suggest that a tannin-based drug may complement the phosphoantigen-based drugs, thereby enhancing the therapeutic potential of {gamma}{delta} T cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The {gamma}{delta} T cell is one of the primary lymphocytes considered to participate in innate immune responses. They are found in virtually all portals of entry into the body where they act as sentinels of the extracellular environment by responding rapidly to tissue damage (1, 2) and pathogens through recognition of conserved pathogen-associated molecular patterns and specific Ags (3, 4, 5). Human {gamma}{delta} T cells can be categorized into two major, functionally distinct populations, V{delta}1 and V{delta}2 T cells, which are characterized by their respective TCR {delta} chain usage. Expression of surface molecules, such as chemokine receptors (6), varies between the {gamma}{delta} T cell subsets, as does their physical locale within the body. V{delta}2 T cells are primarily found in the blood and lymphatic systems, whereas V{delta}1 T cells are typically found in tissues, such as the gut mucosa and skin (7, 8). V{delta}2 T cells are potent antimicrobial and antitumor effector cells (9, 10, 11). V{delta}1 cells have important immunoregulatory functions and, if properly stimulated, can become potent effector cells (12).

Observations that the majority of V{delta}2 T cells proliferate in response to intermediates of the cholesterol synthesis pathway (phosphoantigens) led to clinical trials testing these compounds as potential drugs for the treatment of some cancers and infections (13). Other known {gamma}{delta} T cell agonists, such as bisphosphonates (14) and alkylamines (15), induce a similar response by increasing the endogenous concentration of phosphoantigens. Notably, phosphoantigen-based proliferation requires a costimulatory factor for optimal V{delta}2 T cell expansion, typically IL-2 or IL-15 (16, 17, 18). Though expansion of V{delta}2 T cells in vivo is clearly demonstrated using these new drugs, the benefit of these expanded cells on host defenses is not fully understood due to a minimal or nonexistent response in non-primate animal models (19). Additional caveats for the clinical use of phosphoantigen-based drugs include the requirement for large amounts of agonist (20), induction of rapid cell anergy to treatment (20), and activation of only the V{delta}2 subset, which must then traffic to the tumor or infection to be effective (8, 12, 20, 21).

We initiated a drug discovery effort to identify novel {gamma}{delta} T cell agonists that overcome the shortcomings of phosphoantigen-based drugs. Traditional medicine uses extracts from plants and microbes to treat diseases, many of which have been shown to contain pharmaceutically relevant components. In fact, 47% of the small molecule drugs developed over the last 25 years are either natural products or derived from natural products (22). Phosphostim (Innate Pharma), a {gamma}{delta} T cell agonist in clinical trials, is an example of a drug designed from the structure of naturally occurring phosphoantigens (23, 24). Due to the dominance of pharmaceutically relevant compounds in nature, in an ongoing effort we initiated a screen of >100,000 natural products, including common nutritional supplements, using a FACS-based assay, which measures bovine {gamma}{delta} T cell activation via up-regulation of the IL-2R {alpha} chain (IL-2R{alpha}). Bovine cells were used in the primary screens because, among other reasons, they do not robustly respond to phosphoantigens (25), thus avoiding the detection of these common {gamma}{delta} T cell agonists. In this study we report nonphosphoantigen agonist activity that was observed for both bovine and human {gamma}{delta} T cells in specific plant species including extracts of nonripe Malus domestica (apple) peel, Uncaria tomentosa (Cat’s Claw), Angelica sinensis (Dong Quai), and Funtumia elastica (Yamoa). We traced activity in the apple and Cat’s Claw extracts to the tannin fraction, a class of polyphenols characterized by their capacity to bind proteins. Further analysis identified the bioactive population of tannins as condensed tannins. Detailed dose analyses suggested that superior activity existed in the apple extract. In studies on human cells, apple-derived tannins demonstrated agonist activity for human V{delta}1 and V{delta}2 T cells as well as NK cells. These studies suggest that unique tannin agonists for innate lymphocytes may account, at least in part, for the immunomodulatory properties of some plant extracts. They also suggest that a tannin-based drug may complement the phosphoantigen-based drugs, thereby enhancing the therapeutic potential of {gamma}{delta} T cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Preparation of bovine and human PBMC

Whole blood was collected from 1- to 3-mo-old bull Holstein calves into sodium heparin tubes (BD Biosciences) and from healthy human adult donors with ACD solution B anti-coagulant tubes (BD Biosciences). Leukocytes were separated from whole blood using Histopaque 1077 (Sigma-Aldrich) for bovine cells as previously described (26) and for human cells as per the manufacturer’s instructions. Additionally, bovine RBC were removed by hypotonic lysis. All experiments were performed in accordance with National Institutes of Health guidelines and approved by the Institutional Animal Care and Use Committee and Institutional Review Board of Montana State University (Bozeman, MT).

Preparation of plant extracts

Malus domestica (apple) non-ripe peel (APP3; Apple Poly), Uncaria tomentosa (Cat’s Claw; Raintree Nutrition), Angelica sinensis (Dong Quai; Nature’s Way Products), and Funtumia elastica (Yamoa; NHC) were typically suspended in room temperature water (Cat’s Claw 10 ml/g, Dong Quai 5.0 ml/g, apple peel 8.0 ml/g, and Yamoa 3.0 ml/g), although use of near boiling water had no detectable impact on the preparations, and were agitated for 5 min before centrifugation to remove insoluble particles. Extracts were sterile filtered (0.2 µm) and stored at –80°C until use. Extracts were lyophilized to determine their approximate dry weights: Cat’s Claw 17.6 mg/ml, APP 84.8 mg/ml, Dong Quai 87.0 mg/ml, and Yamoa 32.1 mg/ml.

FACS-based IL-2R{alpha} and CFSE analysis of human and bovine PBMC

PBMC preparations were suspended in X-VIVO 15 medium (Cambrex) at 1 x 106 cells/ml for bovine cells or 2 x 106 cells/ml for human cells. For the measurement of IL-2R{alpha} expression, cells were cultured for 48 h at 37°C, 10% CO2 in the presence or absence of known or test agonists, and the expression of IL-2R{alpha} measured by multicolor FACS (see below). To measure cell proliferation, bovine or human PBMCs were labeled with 2.5 µM CFSE for 5 min in HBSS, washed, and then cultured at 37°C, 10% CO2 in X-VIVO 15 medium in the presence or absence of known or test agonists for 5 days. Human CFSE cultures were typically treated with low-dose human recombinant IL-15 (PeproTech) at the beginning of the culture period. For these cultures, all samples, including controls, received 1.0 ng/ml IL-15. Cell division was quantified by determining the percentage of cells that had divided at least once, based on a 50% or greater reduction in CFSE intensity, using multicolor FACS. Positive control agonists were Con A (0.5 ng/ml; Sigma-Aldrich) with human recombinant IL-2 (1.0 ng/ml; Peprotech) or LPS (10 µg/ml, Escherichia coli O111:B4; Sigma-Aldrich), and negative controls included appropriate buffers diluted in the assay medium.

Cells were analyzed by FACS using a FACSCalibur equipped with an HTS loader (BD Biosciences) or a FACSCanto (BD Biosciences). Bovine cells were stained with IL-2R{alpha} (LCTB2A; VMRD) and/or {gamma}{delta} TCR (GD3.8 (27)). Human cells were stained with IL-2R{alpha} (MEM-181; Serotec), {gamma}{delta} TCR (11F2; BD Biosciences), CD19 (HIB19; BD Biosciences), CD94 (HP-3B1; Beckman Coulter), CD56 (B159; BD Biosciences), CD3 (UCHT1; Beckman Coulter), V{delta}1 TCR (T58.2; Endogen), and/or V{delta}2 TCR (IMMU389; Beckman Coulter) Abs. Abs were directly labeled (FITC, PE, PE-Cy5, PE-Cy7, allophycocyanin, or allophycocyanin-Cy7) or indirectly labeled with goat anti-mouse PE, FITC, or allophycocyanin (Jackson ImmunoResearch Laboratories). Second stage reagents alone were used to determine the level of background staining in the FACS analyses. Only live cells (as determined by FACS light scatter) were included in the analyses.

HPLC analysis of Cat’s Claw

An aqueous extract of Cat’s Claw (1.3 mg in 100 µl) was applied to a reverse phase analytical C18 column (4.6 x 250 mm; Grace Vydac) equilibrated in 0.1% trifluoroacetic acid (Pierce). The extract was eluted with a linear gradient of 0% CH3CN/0.08% trifluoroacetic acid to 100% CH3CN/0.08% trifluoroacetic acid at a flow rate of 1 ml/min. UV absorbance was measured at a wavelength of 254 nm. Fractions were collected at 1-min intervals, dried (SpeedVac; Savant Instruments), and suspended in PBS.

CD69 gene expression

Bovine CD69 gene expression was determined using the Quantigene assay (Panomics) following the manufacturer’s procedure. Briefly, bovine PBMCs (2 x 106 cells/ml) were cultured with the HPLC separated Cat’s Claw fractions (1/20 dilution) for 4 h in complete RPMI 1640 medium, as previously described (3) before lysis with supplied buffer. Lysates were added to prepared 96-well plates containing hybridization probes (provided by the manufacturer) for bovine CD69 mRNA. Presence of hybridized CD69 mRNA was detected with luminescent DNA probes using an LB960 Centro (Berthold Technologies). CD69 gene expression was represented by the fold increase in CD69 signal compared with medium controls.

Separation of tannins from plant extracts

Aqueous extract of Cat’s Claw (10 ml) was treated with 5 ml of polyvinylpolypyrrolidone (PVPP; Sigma-Aldrich) slurry in 0.0625 M HCl at room temperature overnight. The resulting slurry was filtered through a 0.2 µm membrane to remove PVPP and PVPP-bound tannins. The eluent was brought to a pH of 7.2 with NaOH. The tannin-bound PVPP was washed with water before treatment with 0.1 M NaOH for 60 min to remove bound tannins. The solution was again filtered to remove PVPP and brought to a pH of 7.2 with hydrogen chloride. Removal of tannins from Yamoa, Dong Quai, and APP was performed by incubating 1 ml of extract with 100 mg of PVPP for 15 min at 4°C. The unbound fractions were separated from the tannin-bound PVPP by centrifugation. The supernatant fluid was treated with an additional 100 mg of PVPP. This process was repeated a total of five times, reserving samples from each round of depletion.

Tannin fractions from APP, Cat’s Claw, and Dong Quai extracts were subjected to a modified Folin-Ciocalteu assay (28) to measure total phenolics. Briefly, 2 ml of each extract was transferred into a bed volume of 3 ml of PVPP and incubated for 1 h. The nontannin fraction was collected by washing with 10 ml of water. Tannins were eluted with 10 ml of 1.0 M NaOH. Extracts and fractions thereof were serially diluted 1/1 to 1/4050 in water before mixing 500 µl of diluted sample with 250 µl of 1 N Folin-Ciocalteu reagent (Sigma-Aldrich) and 1.25 ml of 20% sodium carbonate. The samples were mixed and incubated at room temperature for 40 min before absorbance was read at 725 nm using a DU800 spectrophotometer (Beckman Coulter). Tannic acid (Sigma-Aldrich) dilutions (40–200 µg/ml) were freshly prepared and used as a standard.

To separate condensed tannins by size, 1.5 ml of APP (~150 mg) was adsorbed to LH-20 resin and washed with 30 ml of water. To elute, 30 ml of 25, 50, 75, and 95% MeOH and 70% acetone were used in succession and the fractions collected. The 95% MeOH and 70% acetone fractions were collected in two 15-ml fractions. Activity was predominantly found in the later fractions, therefore these fractions were used for analyses. All fractions were lyophilized and resuspended in 750 µl of water.

TLR activation and Limulus assays

APP was tested on THP1-Blue CD14 cells (InvivoGen) for TLR agonist activity, according to the manufacturer’s protocol. THP1-Blue CD14 cells express TLRs 1, 2, 4–8, and 10, overexpress CD14, and are transfected with a reporter plasmid containing secreted embryonic alkaline phosphatase (SEAP) under the control of both an NF-{kappa}B and AP-1 inducible promoter. TLR activation was determined by quantifying SEAP activity. Briefly, THP1-Blue CD14 cells at a concentration of 2 x 106 cells/ml were cultured in complete RPMI 1640 medium containing 10% FBS, glucose (4.5 mg/ml), zeocin (200 µg/ml), and blasticidin (10 µg/ml) (all from InvivoGen) followed by PMA (50 ng/ml) treatment for 18 h. PMA was used to differentiate the THP1 cells to induce expression of TLRs. Cells were washed to remove residual PMA and the glucose, zeocin, and blasticidin treatment was discontinued. Cells were stimulated with LPS (a TLR4 agonist), as a positive control, and different concentrations of APP or Cat’s Claw extract in complete RPMI 1640 medium for 24 h at 37°C and 10% CO2. Supernatant fluid was removed and added to Quanti Blue colorimetric assay reagent for 24 h at 37°C and 10% CO2. After 24 h, samples were read at an OD of 655 nm by a VERSAmax tunable microplate reader (Molecular Devices). All samples were run in quadruplicate, from which averages and SDs were determined.

A kinetic turbidimetric Limulus amebocyte lysate (LAL; Associates of Cape Cod) assay was performed on APP as per the manufacturer’s instructions. Briefly, APP was diluted 1/6 in LAL Reagent water and analyzed for endotoxin using LAL Reagent resuspended in Glucashield buffer. LAL agglutination was measured using a ThermoMax Microplate Reader (Molecular Devices). Samples were analyzed in triplicate.

Protease digestion of tannin-associated proteins

APP (42.4 mg) was treated with PVPP (50 mg) and either PBS, chymotrypsin (20 U; Sigma-Aldrich), or trypsin (50,000U; Sigma-Aldrich) for 1 h at 37°C. PVPP was washed, and the tannins eluted with sodium hydroxide.

Alkaline phosphatase inactivation of phosphoantigens

APP extract was passed over a column of polyvinyl spheres containing immobilized alkaline phosphatase (MoBiTec). The phosphoantigen HDMAPP (1-hydroxy-2-methyl-2-buten-4-yl 4-diphosphate; Echelon) or isopentenyl pyrophosphate (Sigma-Aldrich) (both at 500 µg/ml) or APP (42.4 mg/ml) were equilibrated with reaction buffer (final concentrations: 25 mM Tris-HCl, 0.05 mM ZnCl2, 0.5 mM MgCl2 (pH 8.0)) and passed over the column twice. Control samples for were equilibrated with reaction buffer but not passed over the column.

Cell separation using magnetic beads and flow cytometry

PBMCs from bovine and human donors were sorted to create purified cell populations via magnetic bead separation (Miltenyi Biotec), as per the manufacturer’s instructions. Monocytes from bovine and human PBMCs were depleted with magnetic beads conjugated to CD14 mAb using an LD column (Miltenyi Biotec). Purification of human {gamma}{delta} T cells was achieved using either positive or negative sorting techniques. Briefly, human PBMCs were treated with mAbs either to the {gamma}{delta} TCR or to a mixture of mAbs not expressed on {gamma}{delta} T cells (positive and negative sorting, respectively; Miltenyi Biotec). The labeled cells were then linked to magnetic beads and transferred through LS columns (Miltenyi Biotec). The {gamma}{delta} T cells were then collected as the column bound or unbound fractions (positive and negatively sorted {gamma}{delta} T cells, respectively). Bovine {gamma}{delta} T cells were sorted from PBMC by labeling with {gamma}{delta} TCR mAb (GD3.8) and magnetic beads conjugated with anti-mouse Abs (Miltenyi Biotec) as per the manufacturer’s instructions or by using a FACSVantage SE (BD Biosciences) as previously described (26). All sorted cells were cultured under the conditions noted for PBMC cultures, except sorted human {gamma}{delta} T cell cultures, which were cultured between 1 x 105 and 5 x 105 cells/ml.

Purities of magnetically sorted cells were verified by FACS before culture. CD14 depletion was confirmed as <0.5% monocyte contamination using CD14 mAbs (human, UCH-M1; Santa Cruz Biotechnology or bovine, CAM36A; VMRD). {gamma}{delta} T cell purity was determined using {gamma}{delta} TCR mAbs (human, 11F2 or bovine, GD3.8).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Extracts from common dietary plant supplements induce IL-2R{alpha} expression and cell division from {gamma}{delta} T cells in PBMC preparations

Flow cytometry-based assays to determine cell activation (IL-2R{alpha} up-regulation) and proliferation (cell division based on CFSE staining) were used to screen extracts of plants commonly considered having immunomodulatory activity. Bovine {gamma}{delta} T cells were used for these screens to minimize the potential of identifying phosphoantigens because these agonists do not induce robust responses in the bovine model (25). Potent activity was identified in water-soluble extracts from four plant species: nonripe Malus domestica fruit peel (APP), Uncaria tomentosa bark (Cat’s Claw), Angelica sinensis root (Dong Quai), and Funtumia elastica bark (Yamoa) (Fig. 1). Each extract induced IL-2R{alpha} expression on {gamma}{delta} T cells after 48 h in culture and induced some cell division, as detected in a 5-day CFSE assay (Fig. 1). Activity of each extract was specific to {gamma}{delta} T cells and furthermore, nearly all {gamma}{delta} T cells became activated, suggesting activity was not limited to a {gamma}{delta} T cell subset. In cattle there are two major {gamma}{delta} T cell subset classes, the tissue-associated {gamma}{delta} T cell subset, which has similarities to V{delta}1 cells in humans, expresses CD8 and comprises up to 20% of the peripheral {gamma}{delta} T cell population, whereas the predominant blood subset lacks CD8 and expresses the workshop cluster 1 family of glycoproteins (26, 29). Therefore, to confirm that extract activities were not subset specific, additional three-color analyses were performed on 48 h cultures using CD8 (CC58;30) to differentiate the primary bovine {gamma}{delta} T cell subsets. These analyses validated the observations in Fig. 1, demonstrating the agonist activity in APP, Cat’s Claw, Dong Quai, and Yamoa extracts were not limited to one of the primary bovine {gamma}{delta} T cell subsets (data not shown).


Figure 1
View larger version (34K):
[in this window]
[in a new window]

  		FIGURE 1. Plant extracts induce the activation and proliferation of bovine {gamma}{delta} T cells. Freshly collected bovine PBMCs were cultured with Con A/IL-2 (A) or plant extracts (APP, 10.6 µg/ml) (B), Cat’s Claw (44.0 µg/ml) (C), Dong Quai (435.0 µg/ml) (D), or Yamoa (160.5 µg/ml) (E) and analyzed by flow cytometry in IL-2R{alpha} or CFSE two-color FACS assays with fluorescence intensity on a log10 scale. Results for all treatments are representative of a minimum of five independent experiments.

 
Identification of plant tannins as agonists for {gamma}{delta} T cells

As a first step in the identification of the specific agonists in the plant preparations, the Cat’s Claw extract was fractionated on a C-18 HPLC column (Fig. 2A, top). Activity in the collected fractions was measured with bovine PBMCs by cellular expression of surface IL-2R{alpha} or by CD69 gene expression (Fig. 2A, bottom). The broad range of {gamma}{delta} T cell agonist activity observed in the HPLC fractions correlated with the incomplete separation seen in the elution profile, demonstrating a widespread distribution of the active component. A similar result was seen with the APP extract, but initial HPLC separation of the activity in the Yamoa extract failed (data not shown). To gain additional insight into the chemical nature of the active component of Cat’s Claw, we dialyzed the extract against a 50-kDa cut-off membrane. The active fraction was retained (data not shown), suggesting the active component formed large complexes in aqueous solution. Because polyphenols commonly form large, nondialyzable polymeric complexes in aqueous solution (31, 32), and the immunomodulatory activity of many plant extracts resides in the polyphenol fraction (32, 33), we hypothesized that the broad activity profile from samples collected during the HPLC run may be from a group of polyphenolic complexes.


Figure 2
View larger version (18K):
[in this window]
[in a new window]

  		FIGURE 2. Identification of tannins as the agonist component of select plant extracts. A, Cat’s Claw was analyzed by HPLC to characterize the active components. The elution profile from a C-18 HPLC column is represented by absorbance at 254 nm over time in minutes (top). Fractions from the HPLC column were collected at 1-min intervals, and their activity on bovine {gamma}{delta} T cells was analyzed by IL-2R{alpha} cell surface expression (FACS) and CD69 gene expression (bottom). B, Plant extracts were repeatedly treated with PVPP to remove tannins. The resulting tannin-depleted fractions were then assayed for agonist activity by measuring IL-2R{alpha} expression in {gamma}{delta} T cells after 48 h of culture with bovine PBMCs. C, Polyphenol content was determined for APP, Cat’s Claw, and Dong Quai by Folin-Ciocalteu assay. Percentage values represent the fraction of polyphenol comprising the whole extract. The composition of the polyphenol fraction for each extract is further characterized as either tannin ({square}) or nontannin (Figure 2). D, IL-2R{alpha} expression was measured on {gamma}{delta} T cells after incubation with PVPP eluents of APP (36.4 µg/ml) or Cat’s Claw (113.8 µg/ml). Data are representative of at least five experiments.

 
Tannins, a major component of plant polyphenols, are characterized by their ability to bind and precipitate proteins. Therefore, using this peptide-binding nature of tannins, we removed the tannin fraction of the Cat’s Claw sample using the peptide mimetic, PVPP (34). The {gamma}{delta} T cell agonist activity in the Cat’s Claw extract was removed by the PVPP column, as determined by an absence of IL-2R{alpha} up-regulation in bovine {gamma}{delta} T cells treated with the precleared extract (Fig. 2B). APP, Dong Quai, and Yamoa extracts were additionally treated with PVPP to produce tannin-depleted extracts, which were similarly assayed for agonist activity. As shown in Fig. 2B, a single treatment with PVPP removed >90% of the bioactivity in APP. PVPP also removed activity in the Dong Quai extract, although four rounds of depletion were required to remove activity. In contrast, five sequential rounds of PVPP tannin-depletion of the Yamoa extract had minimal impact on its bioactivity (Fig. 2B), suggesting the active component in Yamoa was not a tannin.

To characterize the polyphenol fractions of the putative tannin agonist-containing extracts (APP, Cat’s Claw, and Dong Quai), we performed a series of Folin-Ciocalteu assays (28). These assays were designed to quantify the amount of polyphenols in the entire extracts as well as the tannin and nontannin fractions. The Folin-Ciocalteu assay measures the amount of total polyphenols in a sample. Using tannic acid as a standard for polyphenol weight, we determined the amount of polyphenols contributing to the total dry weight in each of the extracts. Polyphenols contributed to 95% of the total weight of the APP, 56% of the Cat’s Claw, and 4% of the Dong Quai extracts (Fig. 2C). Next, we determined the amount of tannins contributing to the total polyphenols in the extracts by separating tannins and nontannin polyphenols with PVPP before Folin-Ciocalteu assay. Tannins contributed to the majority of polyphenols detected in the APP and Cat’s Claw extracts, 95.1% and 63.7%, respectively. Conversely, the polyphenols in the Dong Quai were predominantly nontannin (90.6%) in character. Interestingly, the Dong Quai extract contained <1% tannins by weight, though PVPP treatment did remove activity (Fig. 2C).

To confirm that tannins in APP, Cat’s Claw, and Dong Quai extracts were the active {gamma}{delta} T cell agonist, the tannin fractions were isolated and assayed for activity. Tannins were separated from each whole extract with PVPP, and then eluted from the PVPP with sodium hdroxide. The resulting tannin fractions were assayed for bioactivity via detection of IL-2R{alpha} expression on bovine {gamma}{delta} T cells. The eluted tannin fraction from Dong Quai demonstrated little bioactivity (data not shown). In contrast, culture with tannin fractions isolated from Cat’s Claw and APP up-regulated IL-2R{alpha} expression on {gamma}{delta} T cells (Fig. 2D), thereby verifying that the {gamma}{delta} T cell agonist in these two extracts was a tannin. Analysis of the PVPP-precipitated tannin fraction via HPLC resulted in a broad elution profile similar to that shown in Fig. 2A (data not shown), confirming the heterogeneous nature of the tannin fraction in these extracts.

We next tested the Cat’s Claw and APP extracts for TLR agonist contaminants, which would potentially confound interpretation of the extract’s activity, using the TLR reporter cell line THP1-Blue CD14 (InvivoGen). This cell line reports TLR1-TLR10 activity (with the exception of TLR3 and TLR9) via NF-{kappa}B/AP-1 transcription of SEAP. No apparent TLR agonist activity was detected in the APP extract, and only minimal TLR agonist activity was detected in the Cat’s Claw extract (Fig. 3A). To confirm that the APP extract did not contain endotoxin at concentrations below detection levels in the THP1-Blue CD14 reporter, we performed a turbidimetric LAL assay. APP was determined to contain <16 endotoxin U/µg. These data demonstrated that the principal activity observed in the tannin fraction of APP, and most likely Cat’s Claw, was not due to TLR agonists.


Figure 3
View larger version (19K):
[in this window]
[in a new window]

  		FIGURE 3. Biochemical analysis of the Cat’s Claw and APP extracts. A, THP1-Blue CD14 cells were cultured with APP (42.4 or 84.8 µg/ml), Cat’s Claw (44 or 88 µg/ml), LPS (10 or 100 µg/ml) at 5000 and 50,000 EU/ml, respectively, or with medium alone. TLR agonist activity was quantified by SEAP secretion. B, APP was incubated with chymotrypsin, trypsin, or PBS in conjunction with PVPP for 1 h. The resulting slurry was washed, and the tannins were eluted from the PVPP. The collected tannins were cultured for 48 h with bovine PBMCs, and then analyzed for IL-2R{alpha} expression on the {gamma}{delta} T cell population. Data represent mean percentage of expression from three different experiments. C, APP condensed tannins adsorbed to LH-20 resin were eluted with increasing concentrations of methanol and a final wash with 70% acetone. The fractions were dried, resuspended in water, and dilutions were cultured with PBMCs for 48 h. IL-2R{alpha} expression on {gamma}{delta} T cells from the cultures was analyzed to determine activity in the condensed tannin fractions.

 
Although PVPP is a fairly specific adsorbent for tannins, proteins, including Haze-Active Proteins 1 and 2 from the apple, associate with tannins and are coadsorbed to PVPP (35). Therefore, to exclude the possibility of protein eliciting the {gamma}{delta} T cell agonist activity, we treated APP with proteases. For these experiments, APP was treated with trypsin or chymotrypsin, adsorbed to PVPP, eluted, and bioassayed via CFSE (data not shown) and IL-2R{alpha} assays (Fig. 3B). Protease treatment of APP had little effect on the agonist activity eluted from subsequent PVPP tannin, demonstrating that the agonist activity was most likely from tannins and not tannin-associated proteins.

Tannins can be classified as either hydrolyzable or condensed. Although structurally different, both types of tannins are adsorbed to PVPP. Therefore we sought to determine to which of these tannin groups the active component of Cat’s Claw and APP belonged. To this end, we treated the PVPP-eluant (total tannins) from Cat’s Claw with hydrogen chloride/mercaptoethanolamine at 90°C for 2 h, a process that hydrolyzes condensed tannins (36). Hydrolysis of the condensed tannins removed {gamma}{delta} T cell activity (data not shown), supporting the concept of condensed tannins as the active component in Cat’s Claw. As an additional test, we sought to confirm the {gamma}{delta} T cell agonist activity of condensed tannins by purifying nondegraded, condensed tannins. We chose to separate condensed tannins from APP because it contained fewer nontannin contaminants and showed no TLR agonist activity. Consequently, we adsorbed APP to an LH-20 column, which specifically adsorbs condensed, but not hydrolyzable tannins. The adsorbed tannins were then eluted with methanol and acetone (37). In addition to separating whole condensed tannins, use of the LH-20 column additionally allows for partial separation of condensed tannins by size. Monomers and dimers preferentially elute in a weaker, 75% MeOH solution, whereas trimers and oligomeric condensed tannins are concentrated in the 95% MeOH and 70% acetone fractions (38). We therefore applied this selective elution method to APP adsorbed to LH-20 and tested the subsequent fractions by IL-2R{alpha} (Fig. 3C) and CFSE (data not shown) assays. We observed {gamma}{delta} T cell activity in the 95% MeOH and acetone fractions, indicating that the predominant {gamma}{delta} T cell agonists in these tannin preparations were trimers or larger (Fig. 3E). We also confirmed that the 95% MeOH and 70% acetone fractions were predominantly larger condensed tannins by TLC (data not shown).

Collectively, these analyses indicated that the {gamma}{delta} T cell agonist activities in Cat’s Claw and APP, and possibly Dong Quai, were due to condensed tannins. In contrast, distinct nontannin {gamma}{delta} T cell agonists existed in the Yamoa extracts.

Effect of APP on human cells

The {gamma}{delta} T cell agonist activity of plant tannins were then characterized on human cells. APP was selected for these analyses because it was more enriched for tannins and demonstrated no detectable TLR agonist activity by either THP1-Blue CD14 reporter cell or Limulus assays. Additionally, APP is predominantly composed of tannins and therefore contains fewer impurities than Cat’s Claw. Activity in the condensed tannin fraction from APP was confirmed in human cells, and this activity was comparable to the total APP preparation (data not shown). As shown in Fig. 4A, APP induced IL-2R{alpha} up-regulation on human {gamma}{delta} T cells, results comparable to those observed in bovine {gamma}{delta} T cells (Fig. 1B). Contrary to the response observed in bovine {gamma}{delta} T cells, wherein a portion of the {gamma}{delta} T cells proliferated in response to APP alone, human {gamma}{delta} T cells required the addition of low-dose IL-15 (1 ng/ml; Fig. 4A) or IL-2 (100 pg/ml; data not shown) to significantly proliferate. This costimulatory requirement for the proliferation of {gamma}{delta} T cells was similar to the well-defined phosphoantigen, HDMAPP (16). However, in contrast to HDMAPP and other phosphoantigen-based agonists, which activate only the V{delta}2 subset of {gamma}{delta} T cells, APP induced IL-2R{alpha} up-regulation and proliferation of both V{delta}1 and V{delta}2 T cells (Fig. 4, B and C). Unlike the {gamma}{delta} T cell-specific proliferative response to APP observed in bovine PBMCs, populations of human non-{gamma}{delta} T cells also responded during culture with the extract. The responding cells included the majority of NK cells and fractions of NKT cells and {alpha}β T cells. There was no significant change in APP-induced B cell proliferation in this assay (Fig. 4D), although B cells have been shown to respond to other tannin preparations (39). Of the responding {alpha}β T cells, naive cells (CD45RO negative cells) were predominant (data not shown). The effective dose range for human cells was ~1–20 µg/ml, with concentrations >20 µg/ml being toxic, which compared closely to the effective dose range for bovine cells of 1–40 µg/ml (data not shown).


Figure 4
View larger version (16K):
[in this window]
[in a new window]

  		FIGURE 4. Characterization of the human PBMC response to APP. Human PBMCs were treated with PBS or APP (4–16 µg/ml) and activation (IL-2R{alpha} expression) or proliferation (CFSE) was measured after 48 h or 5 days of culture, respectively. A, Flow cytometry was used to determine the effect of APP treatment on {gamma}{delta} T cell activation (IL-2R{alpha} expression) (left) and proliferation (CFSE assay) with or without IL-15 (1 ng/ml) (right). Data represent mean values from at least four individuals. B, Activation profiles (IL-2R{alpha} expression) of human V{delta}1 and V{delta}2 T cells treated with APP are shown. Data are representative of three independent experiments. C, Proliferation (CFSE assay) of V{delta}1 and V{delta}2 T cell subsets was measured in response to APP and medium controls (all containing 1 ng/ml IL-15). Values represent the average percentage of V{delta}1 or V{delta}2 T cells that proliferated in samples from four donors. D, Proliferation (CFSE assay) of NK cells (CD3–, CD94+), NKT cells (CD3+, CD94+), {alpha}β T cells (CD3+, {gamma}{delta} TCR–) and B cells (CD3–, CD94–) was measured in IL-15-containing (1 ng/ml) PBMC cultures with either APP or medium alone. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

 
To determine whether {gamma}{delta} T cell proliferation driven by apple tannins was synergistic with the phosphoantigen HDMAPP, human PBMC cultures containing low-dose IL-15 (1 ng/ml) and various concentrations of HDMAPP were additionally cultured with or without 2 µg/ml APP. In the presence of APP, {gamma}{delta} T cells responded nearly as robustly to 1.2 ng/ml HDMAPP as they did to 312 ng/ml HDMAPP alone (Fig. 5A), demonstrating a synergistic effect of APP with phosphoantigen-directed proliferation. Because plants generate phosphoantigens (40), these agonists were a potential contaminant in plant extracts. To address the potential of phosphoantigen activity in the APP, we inactivated potential contaminating phosphoantigens with alkaline phosphatase (23, 41). Alkaline phosphatase treatment of APP had no effect on its bioactivity, whereas the activity of HDMAPP was abolished (Fig. 5B).


Figure 5
View larger version (28K):
[in this window]
[in a new window]

  		FIGURE 5. Human {gamma}{delta} T cell response to the phosphoantigen HDMAPP is augmented by APP. A, Proliferation of CFSE-labeled human PBMCs was measured in response to varying concentrations of HDMAPP with or without 2 µg/ml APP. All cultures, including medium alone, contained 1 ng/ml IL-15. Responses by {gamma}{delta} T cells were determined using {gamma}{delta} TCR mAbs in two-color FACS analyses as described in Materials and Methods. Data are representative of three independent experiments from different donors. B, Proliferative responses of human {gamma}{delta} T cells to APP and HDMAPP preparations that were passed over immobilized alkaline phosphatase (AP), as measured in the two-color FACS-based CFSE assay with 1 ng/ml IL-15. Overlay histograms of gated {gamma}{delta} T cells are presented. Shaded histogram represent buffer-treated samples and solid line histogram represent alkaline phosphatase-treated samples. Data are representative of two independent digestions using PBMCs from three different donors.

 
APP directly activates {gamma}{delta} T cells and primes them to respond to IL-2 or IL-15.

To determine whether {gamma}{delta} T cells responded directly to APP or required signaling through an intermediate cell type, bovine and human PBMC preparations were subjected to various cell separation procedures. Because some tannin preparations impact monocyte cytokine secretion (42), we first compared activation profiles between monocyte-depleted and non-depleted PBMCs from the same bovine or human donor. APP induced equivalent IL-2R{alpha} up-regulation on {gamma}{delta} T cells in both monocyte-containing and -depleted bovine cultures (data not shown) and human cultures (Fig. 6A). To verify that {gamma}{delta} T cells responded directly to APP, human PBMCs were sorted via positive (Fig. 6B) and negative (Fig. 6C) procedures using magnetic beads before culture with APP or medium alone. Cell activation was measured by CD69 and IL-2R{alpha} expression in the positively sorted cells (Fig. 6B). Negatively sorted {gamma}{delta} T cells (Fig. 6C) were assayed for CD69 expression only. Notably, a large fraction of the {gamma}{delta} T cells from the medium controls in both the positively and negatively sorted human {gamma}{delta} T cell cultures were activated (Fig. 6, B and C, medium controls). PBMC cultures from the same donors assayed in parallel did not demonstrate increased baseline activation marker (CD69 and IL-2R{alpha}) expression (data not shown), suggesting the activation was a result of purified human {gamma}{delta} T cells cultured under the conditions used in this experiment. Regardless of the high baseline activation in the purified {gamma}{delta} T cell cultures, there was a considerable increase in activation marker expression (CD69 and IL-2R{alpha}, though changes in CD69 were greater) in APP-treated cultures, demonstrating direct activity for human {gamma}{delta} T cells in the absence of accessory cells.


Figure 6
View larger version (23K):
[in this window]
[in a new window]

  		FIGURE 6. APP induces {gamma}{delta} T cell activation in the absence of accessory cells. Human and bovine {gamma}{delta} T cells were sorted to varying degrees of cell purity and cultured with APP or medium alone for 48 h before FACS analysis of activation marker expression on {gamma}{delta} T cells. A, Monocyte-depleted and nondepleted human PBMCs from four donors were incubated with medium control or APP, and the effect on IL-2R{alpha} expression was determined by FACS. Data represent the concentration of APP inducing maximal activation (4–16 µg/ml). *, p < 0.05 or ***, p < 0.001. B, Positively sorted {gamma}{delta} T cells (91.14%, top, and 83.63%, bottom) obtained from different donors were plated with either APP (12 µg/ml) or medium alone and then were analyzed by FACS for CD69 and IL-2R{alpha} expression. C, Human {gamma}{delta} T cells were collected by negative sort (94.18%) and then cultured with 14 µg/ml APP or medium alone before analysis for CD69 expression. D, IL-2R{alpha} expression was measured on sorted bovine {gamma}{delta} T cells (97.25%) after culture with medium control or APP (31.3 µg/ml).

 
To obtain a purified population of {gamma}{delta} T cells that does not inherently become activated, we sorted bovine {gamma}{delta} T cells via FACS (43) (Fig. 6D) and used these cells as another test to confirm the direct activity of APP on {gamma}{delta} T cells. Bovine peripheral {gamma}{delta} T cells were sorted by flow cytometry to 97.25% purity, treated with PBS or APP, and analyzed for IL-2R{alpha} expression. As shown in Fig. 6D, sorted bovine {gamma}{delta} T cells expressed low IL-2R{alpha} levels following culture in medium alone. The APP-treated, sorted {gamma}{delta} T cell cultures induced IL-2R{alpha} up-regulation to a level comparable to that seen on {gamma}{delta} T cells in a nonsorted culture (see Fig. 1B). As further confirmation of the purified bovine {gamma}{delta} T cell response to APP, cells were positively sorted using magnetic beads (99.1% purity). These cells responded to APP culture in a manner similar to the FACS-sorted bovine {gamma}{delta} T cells; a negligible expression of IL-2R{alpha} was detected in the control population, whereas the APP-treated (10 µg/ml) cells increased IL-2R{alpha} expression greater than 9-fold (data not shown).

Because {gamma}{delta} T cells responded directly to APP with increased expression of activation markers, we next sought to determine whether APP treatment additionally conferred an augmented proliferative status upon the sorted {gamma}{delta} T cell cultures. We first tested monocyte-depleted human and bovine PBMC cultures to determine whether monocytes were required for {gamma}{delta} T cell proliferation in response to APP. Both human and bovine monocyte-depleted cultures demonstrated {gamma}{delta} T cell proliferation when treated with APP (data not shown), demonstrating APP-treated {gamma}{delta} T cells could proliferate in the absence of monocyte-derived growth factors. Next, we tested purified {gamma}{delta} T cell cultures (>96% purity) to determine whether APP-treated {gamma}{delta} T cells could proliferate without the presence of any accessory cells. Sorted bovine {gamma}{delta} T cell cultures were selected for these experiments because high baseline activation was not observed in purified cultures, unlike the sorted human {gamma}{delta} T cell cultures. Initial findings demonstrated that sorted bovine {gamma}{delta} T cells did not proliferate well following APP treatment alone (data not shown), though as shown in Fig. 6C, APP induced IL-2R{alpha} expression. We then tested whether addition of IL-2 or IL-15 would drive cell division in APP pretreated cells (priming assay). To this end, we cultured FACS-sorted, CFSE-labeled bovine {gamma}{delta} T cells for 48 h in culture medium containing either PBS or APP (priming event), washed, and then replaced the medium with either IL-2 (1 ng/ml) or IL-15 (10 ng/ml). After an additional 3 days of culture with the cytokines, the amount of cell division in APP-primed or PBS-treated cells was compared via FACS analysis. As shown in Fig. 7, APP-primed {gamma}{delta} T cells from three different animals responded robustly (77–94% responding cells) to either IL-2 or IL-15, whereas the PBS-primed cells showed minimal response to either cytokine (18–32% responding cells). Thus, APP directly primed {gamma}{delta} T cells to proliferate in response to IL-2 or IL-15 in the absence of accessory cells.


Figure 7
View larger version (35K):
[in this window]
[in a new window]

  		FIGURE 7. APP-primed, purified {gamma}{delta} T cell cultures proliferate in response to IL-2 or IL-15. Sorted bovine {gamma}{delta} T cells were labeled with CFSE and cultured with APP (25 µg/ml) or PBS in culture medium. After 48 h, the medium was replaced with fresh medium containing IL-2 or IL-15, and then cells were cultured for an additional 72–96 h before FACS analysis. All cultures were plated in triplicate. p < 0.001 for all experiments as calculated from the ratio of proliferating to nonproliferating {gamma}{delta} T cells in both of the PBS- and APP-treated controls. Data displayed are pooled from the triplicates collected for each treatment and calf.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
{gamma}{delta} T cells are one of four lymphocyte populations thought to be important in innate immunity, with the other three being NK cells, NKT cells, and B1 B cells. Recently, clinical approaches were developed to expand the number and/or function of human {gamma}{delta} T cells as a potential therapeutic modality for some cancers (44) and to increase innate resistance to infection (7, 10). The current phosphoantigen "drugs" expand the major {gamma}{delta} T cell subset found in blood (V{delta}2), which display potent antitumor cell activity and immune function (13, 45, 46). However, these drugs have no effect on the tissue-predominant human {gamma}{delta} T cell subset (V{delta}1) (47). Thus it is unclear whether phosphoantigen-based drugs will induce the desired effect within tissues, such as the gut mucosa, the skin, and other portals of pathogen entry into the body (8, 12). In this study we show that condensed tannins derived from the unripe peel of the apple fruit are agonists for both human V{delta}1 and V{delta}2 T cells, leading to increased expression of IL-2R{alpha} and cell proliferation. The condensed tannins were active on both human and bovine {gamma}{delta} T cells but showed broader leukocyte subset specificity with human cells. In particular, the human NK cell population was largely responsive to the condensed tannins. Of the two major human {gamma}{delta} T cell subsets, a greater percentage of V{delta}1 T cells responded to the condensed tannins, suggesting that this tissue population might be more responsive to a tannin-based drug preparation in vivo. Though APP was not particularly effective in driving V{delta}2 T cell proliferation unaided, the extract greatly augmented (nearly 300-fold) the proliferative responses of these cells to phosphoantigens and therefore has the potential to be used in combination with phosphoantigens for clinical therapy.

Studies in animals show tissue {gamma}{delta} T cells not only serve as effector cells, but also participate in immunoregulation (7), tissue homeostasis (2), and epithelial wound repair (48). Increasing these functions would directly and indirectly enhance host innate and downstream adaptive immune responses. In this study, we found that the tissue-associated {gamma}{delta} T cells in both human (V{delta}1 T cells) and bovine (CD8+ {gamma}{delta} T cells) respond to APP. Likewise, Akiyama et al. (49) show that mouse mucosal {gamma}{delta} T cells expand in response to apple-condensed tannins in vivo, which we have confirmed with in vitro assays (M.A. Jutila, unpublished results). Because these responses to tannins are conserved in various species, tannin recognition may represent an ancient mechanism used by these cells to sense and respond to their environment. One would expect plant tannin-based agonists to be variably represented in the diet of each of these animals, including humans. Ruminants are exposed to a very high load of plant derived tannins and this may account for, in part, the very large numbers of {gamma}{delta} T cells seen in these animals.

The best-defined agonists that drive {gamma}{delta} T cell expansion are phosphoantigens, but they are specific to human and non-human primate cells (19) making in vivo studies difficult. Limited studies in non-human primates demonstrate proof-of-principle that {gamma}{delta} T cells expand in vivo (20), but efficacy studies have not been reported in these animals. To circumvent the difficulties inherent in using non-human primates for in vivo studies, efficacy studies have been performed in SCID mice reconstituted with phosphoantigen-expanded human V{delta}2 T cells. These studies demonstrate phosphoantigen-expanded V{delta}2 T cell effector function against tumors (45) and bacteria (9). However, challenge studies in immunocompetent animals have not been performed. Until these studies are performed, understanding the effectiveness of phosphoantigen-expanded {gamma}{delta} T cells in a clinical setting will remain unresolved.

Because the {gamma}{delta} T cell response to condensed tannins is conserved in a wide range of species, there are multiple models available to test the in vivo efficacy of these condensed tannins in infectious disease, wound repair/healing, and cancers. Limited work already published on the extracts examined in this study support the likelihood for success in these experiments. For instance, Cat’s Claw is a traditional South American medicine used to treat a variety of illnesses including allergies, cancer, colitis, dysentery, and urinary tract infection (50). Interestingly, these illnesses are commonly associated with {gamma}{delta} T cell regulatory activities (7, 51, 52, 53, 54), suggesting the described immunostimulatory properties of the Cat’s Claw plant could be partially due to the activation of {gamma}{delta} T cells. Preparations containing the apple-condensed tannins we described are also recognized for similar immunostimulatory properties (49, 55, 56, 57). These observations on the immunological effects of Cat’s Claw and apple tannins support the prospect for condensed tannins to be used as a therapy to increase {gamma}{delta} T cell function in innate immunity and wound healing.

The results of this study may also provide another explanation for the effect of tea consumption on human {gamma}{delta} T cell activation. Kamath et al. (58) demonstrate an in vivo {gamma}{delta} T cell priming event corresponding to tea intake. After 14 days of tea consumption, the study shows that {gamma}{delta} T cells become more responsive to in vitro phosphoantigen-based stimulation. They attribute the effect of tea to L-theonine, which is converted to alkylamines by the liver. The alkylamines then purportedly drive the expansion/priming of the phosphoantigen-responsive {gamma}{delta} T cells. However, tea is a rich source of immunomodulatory tannins, and we have found that hot water extracts of black tea induce bovine {gamma}{delta} T cell responses (M.A. Jutila, unpublished observations). The tannin fraction of tea may prime human {gamma}{delta} T cells, in a manner akin to the APP response described in this experiment (Fig. 5), making them more responsive to in vitro phosphoantigen stimulation. Thus, tannins derived from tea may account, at least in part, for the priming effects on {gamma}{delta} T cells observed in vivo following tea consumption.

A major implication of these observations is that a tannin-based drug may enhance the effectiveness of current phosphoantigen-based drugs and other approaches to increase innate responses by lymphocytes. Enhancement of phosphoantigen responses may be of particular benefit to individuals afflicted with hyporesponsive V{delta}2 populations due to either repeat phosphoantigen treatment (20) or persistent infection (59, 60, 61). As shown in Fig. 5A, APP improves the proliferation of HDMAPP-stimulated {gamma}{delta} T cells. Therefore, in these settings, it may be possible to increase the number of expanding V{delta}2 cells by coadministering a tannin preparation. The effects of a tannin agonist would likely extend to other human lymphocytes as well. As shown in Fig. 5, NK cells were actually more responsive to APP in the CFSE assay than {gamma}{delta} T cells. NK cells are important in a variety of innate functions, including roles as potent antiviral and antitumor cells as well as producers of IFN-{gamma}. Currently, attempts to expand NK cells for clinical benefit focus on cytokine-based expansion and are therefore troubled with toxicity due to non-target cell activation (62, 63). Our in vitro data suggest the potential for tannin-derived agonists to aid, in a manner akin to phosphoantigen/IL-2 stimulation, the cytokine-based expansion of this innate cell population. A fraction of {alpha}β T cells also responded within our assays. Analysis of CD45RO expression suggested that the majority of the responding {alpha}β T cells were, surprisingly, naive T cells. This suggests that some primary Ag-driven {alpha}β T cell responses might be enhanced by these tannins, but this requires further testing.

A tannin-based drug has the potential to not only be used therapeutically, but prophylactically as well. A concern with the prophylactic use of any immunomodulatory compound is the induction of chronic inflammation. Current phosphoantigen-based drugs lead to overt cytokine release in humans (64, 65) including the proinflammatory cytokines TNF-{alpha} and IFN-{gamma}, which would contribute to systemic inflammation in the patient. In a concurrent study, we found that the tannin preparation we tested up-regulated CD11b on {gamma}{delta} T cells, but importantly, did not alter transcript levels for TNF-{alpha}. However, GM-CSF and MIP1{alpha} gene transcription was induced (43). Thus, activation occurs following tannin treatment, but the response appears to be far more subtle than that observed with phosphoantigens. Our data suggests that under a prophylactic treatment regimen, tannin agonists prime but do not overtly activate cells. Upon introduction of an insult, such as exposure to a mucosal pathogen, we postulate that "primed" {gamma}{delta} T cells would respond faster and more robustly to secondary agonists such as pathogen-associated molecular patterns and cytokines secreted from other innate cells, such as macrophages.

The chemical nature of the apple tannins responsible for the agonist activity for {gamma}{delta} T cell is not defined. Most plants produce tannins, but their chemical characteristics vary considerably between different species. Though we are only now beginning to catalog these results, it is clear that not all plant tannins are {gamma}{delta} T cell agonists. In screens of common dietary supplements, potent agonist activity was detected in extracts of the plants described in this study, but the majority of plants tested contained minimal to no {gamma}{delta} T cell agonist activity. In our ongoing drug discovery efforts, we are characterizing eight plant extracts with potent non-tannin {gamma}{delta} T cell agonist activity (A. Palecanda, and M. Jutila, unpublished observations). Yamoa is an example of one such plant; although it produces complex tannins (data not shown), its activity for {gamma}{delta} T cells resides in its non-tannin fraction (Fig. 3A).

Condensed tannins range from simple monomers to highly complex oligomers (33). The {gamma}{delta} T cell agonist described in this experiment elutes with the larger condensed tannin population (Fig. 3E). Large condensed tannins are prohibitive to synthesize, but may be amendable to bulk purification procedures. Techniques of bulk tannin purification previously developed by the food industry may be adaptable to purify the tannin complex involved in these responses (66, 67). Using these tannin isolation procedures for purification and characterization of the minimal tannin complex from APP is a primary focus in further evaluating the potential for use of these compounds as therapeutic and/or prophylactic agents.

Questions remain regarding the specific mechanisms of tannin activity on leukocytes. Originally defined as low-affinity and nonspecific protein binding complexes with antioxidant activity, tannins are increasingly portrayed as having high-affinity counter receptors (68, 69). An example of an immunologically relevant tannin-protein specific interaction is found in tannic acid. Tannic acid is able to block the chemokine CXCL12 from binding to its receptor, CXCR4. The action of tannic acid is specific to CXCL12/CXCR4 given that other chemokines/chemoattractants are not effected by this tannin preparation (70). Furthermore, apple tannins block Fc{epsilon}R1/IgE binding (55) and prevent epidermal growth factor signaling, presumably through physically blocking the receptor (57). Similar polyphenols from tea bind CD11b on monocytes, which we recently found for APP as well (43). Characterizing a potential receptor mediated response triggered by APP in {gamma}{delta} T cells was well beyond the intent of this initial study. However, the data presented in this study are consistent with APP acting through one, or perhaps a restricted number of, receptors on the T cell and not through a nonspecific mechanism, such as antioxidant activity. Assuming the active agonist represents a fraction of the total crude material, activity is likely within nanogram per milliliter levels. This level of activity is consistent with signaling through a specific receptor. Studies describing the specific binding of tannic acid to CXCR4 show this interaction competitively inhibits CXCL12 binding at concentrations (IC50, 360 ng/ml) (70) similar to those we predict for the active component of APP. The restrictive pattern of gene regulation induced by APP in {gamma}{delta} T cells, selective up-regulation of IL-2R{alpha}, CD69, MIP1{alpha}, and GM-CSF shown in this study and by Graff and Jutila (43), is also consistent with a receptor-mediated event. We are currently investigating the receptors that may be involved in these responses.

The effective dose range of crude APP required to induce IL-2R{alpha} on {gamma}{delta} T cells was quite limited (1–40 µg/ml for bovine cells and 1–20 µg/ml for human cells), but this was due to toxicity (induction of apoptosis) of the preparations at higher concentrations under the assay conditions tested. This level of toxicity is consistent with other tannin preparations (70). These in vitro data suggest a relatively narrow therapeutic and nontoxic range for tannins. However, in vivo studies in rats and mice demonstrate high-dose oral administration without toxic effects. Apple tannins provided to mice ad libitium in their drinking water at 1.0% w/v lead to expansion of {gamma}{delta} T cells in the gut mucosa, but this dose does not induce obvious toxicity (49). Furthermore, when APP is given orally to rats at concentrations up to 2000 mg/kg, all animals survive, gain body weight, and overt inflammation of the gut mucosa or any other organ is not observed over a 14-day period (71). The reason such high doses of APP administered orally are not toxic may be because the gut appears to regulate the uptake of orally administered tannins and prevents toxic levels from making it to the bloodstream. Specifically, plasma uptake of tannins in rats plateaus at 10.2 µg/ml (1000 mg/kg oral dose) and does not increase when rats are fed 2000 mg/kg of a tannin preparation (72). This suggests oral administration may naturally prevent overdose, and regulate tannin concentrations in the plasma at optimal mitogenic concentrations.

In summary, although phosphoantigens are potent stimulators of human {gamma}{delta} T cells, they possess limitations that may prevent their reliable expansion of {gamma}{delta} T cell populations for potential treatment of a variety of diseases. Therefore, the search for additional {gamma}{delta} T cell agonists capable of improving phosphoantigen stimulation of V{delta}2 T cells as well as agonists capable of expanding the V{delta}1 subset is warranted. Our results indicate tannins derived from plants tissues, such as Cat’s Claw bark and nonripe apple peel, represent distinct and complementary agonists to phosphoantigens. Therefore, tannin-based therapeutics can potentially address some shortcomings of phosphoantigen-based drugs by functioning as stand-alone drugs or drugs used in combination with other immunomodulatory compounds.


   Acknowledgments
 
We thank Matt Calverley for preparing the THP1-Blue CD14 TLR assays, Evelyn Benson for performing the Limulus assays, and Kathryn Crist, Kirk Lubick, and Jill Graff for critical review and several helpful suggestions.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
M. A. Jutila holds shares in LigoCyte Pharmaceuticals, Inc., which together with Montana State University, holds a National Institutes of Health contract that partially funded this work. All other Montana State University authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by funding in whole or in part from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, and by Contract HHSN266200400009/N01-AI40009 from the Department of Health and Human Services. This work is also supported by Contract 2000-04446 from the U.S. Department of Agriculture Initiative Future Agriculture and Food Safety, and National Research Initiative. LigoCyte Pharmaceuticals, Inc., together with Montana State University, also holds a National Institutes of Health contract that partially funded this work. Back

2 Address correspondence and reprint requests to Dr. Mark A. Jutila, Veterinary Molecular Biology, Molecular Biosciences Building, Montana State University, 960 Technology Boulevard, Bozeman, MT 59718. E-mail address: uvsmj{at}montana.edu Back

3 Abbreviations used in this paper: APP, apple polyphenol; PVPP, polyvinylpolypyrrolidone; SEAP, secreted embryonic alkaline phosphatase; LAL, Limulus amebocyte lysate. Back

Received for publication July 30, 2007. Accepted for publication August 30, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Chen, Y., K. Chou, E. Fuchs, W. L. Havran, R. Boismenu. 2002. Protection of the intestinal mucosa by intraepithelial {gamma}{delta} T cells. Proc. Natl. Acad. Sci. USA 99: 14338-14343. [Abstract/Free Full Text]
  2. Sharp, L. L., J. M. Jameson, G. Cauvi, W. L. Havran. 2005. Dendritic epidermal T cells regulate skin homeostasis through local production of insulin-like growth factor 1. Nat. Immunol. 6: 73-79. [Medline]
  3. Hedges, J. F., K. J. Lubick, M. A. Jutila. 2005. {gamma}{delta} T cells respond directly to pathogen-associated molecular patterns. J. Immunol. 174: 6045-6053. [Abstract/Free Full Text]
  4. Deetz, C. O., A. M. Hebbeler, N. A. Propp, C. Cairo, I. Tikhonov, C. D. Pauza. 2006. Gamma interferon secretion by human V{gamma}2V{delta}2 T cells after stimulation with antibody against the T-cell receptor plus the Toll-Like receptor 2 agonist Pam3Cys. Infect. Immun. 74: 4505-4511. [Abstract/Free Full Text]
  5. Lubick, K., M. A. Jutila. 2006. LTA recognition by bovine {gamma}{delta} T cells involves CD36. J. Leukoc. Biol. 79: 1268-1270. [Abstract/Free Full Text]
  6. Kabelitz, D., D. Wesch. 2003. Features and functions of {gamma}{delta} T lymphocytes: focus on chemokines and their receptors. Crit. Rev. Immunol. 23: 339-370. [Medline]
  7. Girardi, M.. 2006. Immunosurveillance and immunoregulation by {gamma}{delta} T cells. J. Invest. Dermatol. 126: 25-31. [Medline]
  8. Deusch, K., F. Luling, K. Reich, M. Classen, H. Wagner, K. Pfeffer. 1991. A major fraction of human intraepithelial lymphocytes simultaneously expresses the {gamma}/{delta} T cell receptor, the CD8 accessory molecule and preferentially uses the V{delta}1 gene segment. Eur. J. Immunol. 21: 1053-1059. [Medline]
  9. Wang, L., A. Kamath, H. Das, L. Li, J. F. Bukowski. 2001. Antibacterial effect of human V{gamma}2V{delta}2 T cells in vivo. J. Clin. Invest. 108: 1349-1357. [Medline]
  10. Poccia, F., C. Agrati, F. Martini, G. Mejia, M. Wallace, M. Malkovsky. 2005. V{gamma}9V{delta}2 T cell-mediated non-cytolytic antiviral mechanisms and their potential for cell-based therapy. Immunol. Lett. 100: 14-20. [Medline]
  11. Lamb, L. S., Jr, R. D. Lopez. 2005. {gamma}{delta} T cells: a new frontier for immunotherapy?. Biol. Blood Marrow Transplant 11: 161-168. [Medline]
  12. Ebert, L. M., S. Meuter, B. Moser. 2006. Homing and function of human skin {gamma}{delta} T cells and NK cells: relevance for tumor surveillance. J. Immunol. 176: 4331-4336. [Abstract/Free Full Text]
  13. Viey, E., G. Fromont, B. Escudier, Y. Morel, S. Da Rocha, S. Chouaib, A. Caignard. 2005. Phosphostim-activated {gamma}{delta} T cells kill autologous metastatic renal cell carcinoma. J. Immunol. 174: 1338-1347. [Abstract/Free Full Text]
  14. Dunford, J. E., K. Thompson, F. P. Coxon, S. P. Luckman, F. M. Hahn, C. D. Poulter, F. H. Ebetino, M. J. Rogers. 2001. Structure-activity relationships for inhibition of farnesyl diphosphate synthase in vitro and inhibition of bone resorption in vivo by nitrogen-containing bisphosphonates. J. Pharmacol. Exp. Ther. 296: 235-242. [Abstract/Free Full Text]
  15. Thompson, K., J. Rojas-Navea, M. J. Rogers. 2006. Alkylamines cause V{gamma}9V{delta}2 T-cell activation and proliferation by inhibiting the mevalonate pathway. Blood 107: 651-654. [Abstract/Free Full Text]
  16. Amslinger, S., S. Hecht, F. Rohdich, W. Eisenreich, P. Adam, A. Bacher, S. Bauer. 2007. Stimulation of V{gamma}9/V{delta}2 T-lymphocyte proliferation by the isoprenoid precursor, (E)-1-hydroxy-2-methyl-but-2-enyl 4-diphosphate. Immunobiology 212: 47-55. [Medline]
  17. Sato, K., S. Kimura, H. Segawa, A. Yokota, S. Matsumoto, J. Kuroda, M. Nogawa, T. Yuasa, Y. Kiyono, H. Wada, T. Maekawa. 2005. Cytotoxic effects of {gamma}{delta} T cells expanded ex vivo by a third generation bisphosphonate for cancer immunotherapy. Int. J. Cancer 116: 94-99. [Medline]
  18. Garcia, V. E., D. Jullien, M. Song, K. Uyemura, K. Shuai, C. T. Morita, R. L. Modlin. 1998. IL-15 enhances the response of human {gamma}{delta} T cells to nonpeptide [correction of nonpetide] microbial antigens. J. Immunol. 160: 4322-4329. [Abstract/Free Full Text]
  19. Wang, H., H. K. Lee, J. F. Bukowski, H. Li, R. A. Mariuzza, Z. W. Chen, K. H. Nam, C. T. Morita. 2003. Conservation of nonpeptide antigen recognition by rhesus monkey V{gamma}2V{delta}2 T cells. J. Immunol. 170: 3696-3706. [Abstract/Free Full Text]
  20. Sicard, H., S. Ingoure, B. Luciani, C. Serraz, J. J. Fournie, M. Bonneville, J. Tiollier, F. Romagne. 2005. In vivo immunomanipulation of V{gamma}9V{delta}2 T cells with a synthetic phosphoantigen in a preclinical nonhuman primate model. J. Immunol. 175: 5471-5480. [Abstract/Free Full Text]
  21. Kunzmann, V., E. Bauer, M. Wilhelm. 1999. {gamma}/{delta} T-cell stimulation by pamidronate. N. Engl. J. Med. 340: 737-738. [Free Full Text]
  22. Newman, D. J., G. M. Cragg. 2007. Natural products as sources of new drugs over the last 25 years. J. Nat. Prod. 70: 461-477. [Medline]
  23. Tanaka, Y., S. Sano, E. Nieves, G. De Libero, D. Rosa, R. L. Modlin, M. B. Brenner, B. R. Bloom, C. T. Morita. 1994. Nonpeptide ligands for human {gamma}{delta} T cells. Proc. Natl. Acad. Sci. USA 91: 8175-8179. [Abstract/Free Full Text]
  24. Poupot, M., J. J. Fournie. 2004. Non-peptide antigens activating human V{gamma}9/V{delta}2 T lymphocytes. Immunol. Lett. 95: 129-138. [Medline]
  25. Vesosky, B., O. C. Turner, J. Turner, I. M. Orme. 2004. Gamma interferon production by bovine {gamma}{delta} T cells following stimulation with mycobacterial mycolylarabinogalactan peptidoglycan. Infect. Immun. 72: 4612-4618. [Abstract/Free Full Text]
  26. Hedges, J. F., D. Cockrell, L. Jackiw, N. Meissner, M. A. Jutila. 2003. Differential mRNA expression in circulating {gamma}{delta} T lymphocyte subsets defines unique tissue-specific functions. J. Leukoc. Biol. 73: 306-314. [Abstract/Free Full Text]
  27. Wilson, E., B. Walcheck, W. C. Davis, M. A. Jutila. 1998. Preferential tissue localization of bovine {gamma}{delta} T cell subsets defined by anti-T cell receptor for antigen antibodies. Immunol. Lett. 64: 39-44. [Medline]
  28. Singleton, V. L., J. A. Rossi, Jr. 1965. Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. Am. J. Enol. Viticulture 16: 144-158. [Abstract/Free Full Text]
  29. MacHugh, N. D., J. K. Mburu, M. J. Carol, C. R. Wyatt, J. A. Orden, W. C. Davis. 1997. Identification of two distinct subsets of bovine {gamma}{delta} T cells with unique cell surface phenotype and tissue distribution. Immunology 92: 340-345. [Medline]
  30. MacHugh, N. D., A. Bensaid, C. J. Howard, W. C. Davis, W. I. Morrison. 1991. Analysis of the reactivity of anti-bovine CD8 monoclonal antibodies with cloned T cell lines and mouse L-cells transfected with bovine CD8. Vet. Immunol. Immunopathol. 27: 169-172. [Medline]
  31. Rohrbach, M. S., T. J. Kreofsky, B. G. Chan. 1990. Structural determinants of the platelet agonist activity of cotton bract condensed tannin. Environ. Res. 52: 199-209. [Medline]
  32. Chan, W. S., S. Chowdhry, T. Chang, R. R. Kew. 1999. Initial characterization of the complement activating compounds in extracts of smokeless tobacco. Immunobiology 201: 64-73. [Medline]
  33. Okuda, T.. 2005. Systematics and health effects of chemically distinct tannins in medicinal plants. Phytochemistry 66: 2012-2031. [Medline]
  34. Toth, G. B., H. Pavia. 2001. Removal of dissolved brown algal phlorotannins using insoluble polyvinylpolypyrrolidone (PVPP). J. Chem. Ecol. 27: 1899-1910. [Medline]
  35. Wu, L. C., K. J. Siebert. 2002. Characterization of haze-active proteins in apple juice. J. Agric. Food Chem. 50: 3828-3834. [Medline]
  36. Selga, A., J. L. Torres. 2005. Efficient preparation of catechin thio conjugates by one step extraction/depolymerization of pine (Pinus pinaster) bark procyanidins. J. Agric. Food Chem. 53: 7760-7765. [Medline]
  37. Strumeyer, D. H., M. J. Malin. 1975. Condensed tannins in grain sorghum: isolation, fractionation, and characterization. J. Agric. Food Chem. 23: 909-914. [Medline]
  38. Taylor, A. W., E. Barofsky, J. A. Kennedy, M. L. Deinzer. 2003. Hop (Humulus lupulus L.) proanthocyanidins characterized by mass spectrometry, acid catalysis, and gel permeation chromatography. J. Agric. Food Chem. 51: 4101-4110. [Medline]
  39. Kenny, T. P., C. L. Keen, H. H. Schmitz, M. E. Gershwin. 2007. Immune effects of cocoa procyanidin oligomers on peripheral blood mononuclear cells. Exp. Biol. Med. 232: 293-300. [Abstract/Free Full Text]
  40. Eisenreich, W., A. Bacher, D. Arigoni, F. Rohdich. 2004. Biosynthesis of isoprenoids via the non-mevalonate pathway. Cell Mol. Life Sci. 61: 1401-1426. [Medline]
  41. Fischer, S., A. Scheffler, D. Kabelitz. 1996. Activation of human {gamma}{delta} T-cells by heat-treated mistletoe plant extracts. Immunol. Lett. 52: 69-72. [Medline]
  42. Kolodziej, H., A. F. Kiderlen. 2005. Antileishmanial activity and immune modulatory effects of tannins and related compounds on Leishmania parasitised RAW 264.7 cells. Phytochemistry 66: 2056-2071. [Medline]
  43. Graff, J. C., M. A. Jutila. 2007. Differential regulation of CD11b on {gamma}{delta} T cells and monocytes in response to unripe apple polyphenols. J. Leukoc. Biol. 82: 603-607. [Abstract/Free Full Text]
  44. Kabelitz, D., D. Wesch, W. He. 2007. Perspectives of {gamma}{delta} T cells in tumor immunology. Cancer Res. 67: 5-8. [Abstract/Free Full Text]
  45. Kabelitz, D., D. Wesch, E. Pitters, M. Zoller. 2004. Characterization of tumor reactivity of human V{gamma}9V{delta}2 {gamma}{delta} T cells in vitro and in SCID mice in vivo. J. Immunol. 173: 6767-6776. [Abstract/Free Full Text]
  46. Poccia, F., L. Battistini, B. Cipriani, G. Mancino, F. Martini, M. L. Gougeon, V. Colizzi. 1999. Phosphoantigen-reactive V{gamma}9V{delta}2 T lymphocytes suppress in vitro human immunodeficiency virus type 1 replication by cell-released antiviral factors including CC chemokines. J. Infect. Dis. 180: 858-861. [Medline]
  47. Tanaka, Y., C. T. Morita, Y. Tanaka, E. Nieves, M. B. Brenner, B. R. Bloom. 1995. Natural and synthetic non-peptide antigens recognized by human {gamma}{delta} T cells. Nature 375: 155-158. [Medline]
  48. Jameson, J. M., G. Cauvi, L. L. Sharp, D. A. Witherden, W. L. Havran. 2005. {gamma}{delta} T cell-induced hyaluronan production by epithelial cells regulates inflammation. J. Exp. Med. 201: 1269-1279. [Abstract/Free Full Text]
  49. Akiyama, H., Y. Sato, T. Watanabe, M. H. Nagaoka, Y. Yoshioka, T. Shoji, T. Kanda, K. Yamada, M. Totsuka, R. Teshima, J. Sawada, et al 2005. Dietary unripe apple polyphenol inhibits the development of food allergies in murine models. FEBS Lett. 579: 4485-4491. [Medline]
  50. Heitzman, M. E., C. C. Neto, E. Winiarz, A. J. Vaisberg, G. B. Hammond. 2005. Ethnobotany, phytochemistry and pharmacology of Uncaria (Rubiaceae). Phytochemistry 66: 5-29. [Medline]
  51. Tsuchiya, T., S. Fukuda, H. Hamada, A. Nakamura, Y. Kohama, H. Ishikawa, K. Tsujikawa, H. Yamamoto. 2003. Role of {gamma}{delta} T cells in the inflammatory response of experimental colitis mice. J. Immunol. 171: 5507-5513. [Abstract/Free Full Text]
  52. Jonasson, R., A. Johannisson, M. Jacobson, C. Fellstrom, M. Jensen-Waern. 2004. Differences in lymphocyte subpopulations and cell counts before and after experimentally induced swine dysentery. J. Med. Microbiol. 53: 267-272. [Abstract/Free Full Text]
  53. Cairo, C., E. Arabito, F. Landi, A. Casati, E. Brunetti, G. Mancino, E. Galli. 2005. Analysis of circulating {gamma}{delta} T cells in children affected by IgE-associated and non-IgE-associated allergic atopic eczema/dermatitis syndrome. Clin. Exp. Immunol. 141: 116-121. [Medline]
  54. Jones-Carson, J., E. Balish, D. T. Uehling. 1999. Susceptibility of immunodeficient gene-knockout mice to urinary tract infection. J. Urol. 161: 338-341. [Medline]
  55. Tokura, T., N. Nakano, T. Ito, H. Matsuda, Y. Nagasako-Akazome, T. Kanda, M. Ikeda, K. Okumura, H. Ogawa, C. Nishiyama. 2005. Inhibitory effect of polyphenol-enriched apple extracts on mast cell degranulation in vitro targeting the binding between IgE and Fc{epsilon}RI. Biosci. Biotechnol. Biochem. 69: 1974-1977. [Medline]
  56. Enomoto, T., Y. Nagasako-Akazome, T. Kanda, M. Ikeda, Y. Dake. 2006. Clinical effects of apple polyphenols on persistent allergic rhinitis: a randomized double-blind placebo-controlled parallel arm study. J. Investig. Allergol. Clin. Immunol. 16: 283-289. [Medline]
  57. Kern, M., Z. Tjaden, Y. Ngiewih, N. Puppel, F. Will, H. Dietrich, G. Pahlke, D. Marko. 2005. Inhibitors of the epidermal growth factor receptor in apple juice extract. Mol. Nutr. Food Res. 49: 317-328. [Medline]
  58. Kamath, A. B., L. Wang, H. Das, L. Li, V. N. Reinhold, J. F. Bukowski. 2003. Antigens in tea-beverage prime human V{gamma}2V{delta}2 T cells in vitro and in vivo for memory and nonmemory antibacterial cytokine responses. Proc. Natl. Acad. Sci. USA 100: 6009-6014. [Abstract/Free Full Text]
  59. Poccia, F., S. Boullier, H. Lecoeur, M. Cochet, Y. Poquet, V. Colizzi, J. J. Fournie, M. L. Gougeon. 1996. Peripheral V{gamma}9/V{delta}2 T cell deletion and anergy to nonpeptidic mycobacterial antigens in asymptomatic HIV-1-infected persons. J. Immunol. 157: 449-461. [Abstract]
  60. Wilhelm, M., V. Kunzmann, S. Eckstein, P. Reimer, F. Weissinger, T. Ruediger, H. P. Tony. 2003. {gamma}{delta} T cells for immune therapy of patients with lymphoid malignancies. Blood 102: 200-206. [Abstract/Free Full Text]
  61. Li, B., M. D. Rossman, T. Imir, A. F. Oner-Eyuboglu, C. W. Lee, R. Biancaniello, S. R. Carding. 1996. Disease-specific changes in {gamma}{delta} T cell repertoire and function in patients with pulmonary tuberculosis. J. Immunol. 157: 4222-4229. [Abstract]
  62. Duvic, M., M. L. Sherman, G. S. Wood, T. M. Kuzel, E. Olsen, F. Foss, R. J. Laliberte, J. L. Ryan, K. Zonno, A. H. Rook. 2006. A phase II open-label study of recombinant human interleukin-12 in patients with stage IA, IB, or IIA mycosis fungoides. J. Am. Acad. Dermatol. 55: 807-813. [Medline]
  63. Wang, G., M. Tschoi, R. Spolski, Y. Lou, K. Ozaki, C. Feng, G. Kim, W. J. Leonard, P. Hwu. 2003. In vivo antitumor activity of interleukin 21 mediated by natural killer cells. Cancer Res. 63: 9016-9022. [Abstract/Free Full Text]
  64. Conti, L., R. Casetti, M. Cardone, B. Varano, A. Martino, F. Belardelli, F. Poccia, S. Gessani. 2005. Reciprocal activating interaction between dendritic cells and pamidronate-stimulated {gamma}{delta} T cells: role of CD86 and inflammatory cytokines. J. Immunol. 174: 252-260. [Abstract/Free Full Text]
  65. Casetti, R., G. Perretta, A. Taglioni, M. Mattei, V. Colizzi, F. Dieli, G. D’Offizi, M. Malkovsky, F. Poccia. 2005. Drug-induced expansion and differentiation of V{gamma}9V{delta}2 T cells in vivo: the role of exogenous IL-2. J. Immunol. 175: 1593-1598. [Abstract/Free Full Text]
  66. Laborde, B., V. Moine-Ledoux, T. Richard, C. Saucier, D. Dubourdieu, J. P. Monti. 2006. PVPP-polyphenol complexes: a molecular approach. J. Agric. Food Chem. 54: 4383-4389. [Medline]
  67. Saucier, C., M. Jourdes, Y. Glories, S. Quideau. 2006. Extraction, detection, and quantification of flavano-ellagitannins and ethylvescalagin in a Bordeaux red wine aged in oak barrels. J. Agric. Food Chem. 54: 7349-7354. [Medline]
  68. Hagerman, A. E., L. G. Butler. 1981. The specificity of proanthocyanidin-protein interactions. J. Biol. Chem. 256: 4494-4497. [Abstract/Free Full Text]
  69. Frazier, R. A., A. Papadopoulou, I. Mueller-Harvey, D. Kissoon, R. J. Green. 2003. Probing protein-tannin interactions by isothermal titration microcalorimetry. J. Agric. Food Chem. 51: 5189-5195. [Medline]
  70. Chen, X., J. A. Beutler, T. G. McCloud, A. Loehfelm, L. Yang, H. F. Dong, O. Y. Chertov, R. Salcedo, J. J. Oppenheim, O. M. Howard. 2003. Tannic acid is an inhibitor of CXCL12 (SDF-1{alpha})/CXCR4 with antiangiogenic activity. Clin. Cancer Res. 9: 3115-3123. [Abstract/Free Full Text]
  71. Shoji, T., Y. Akazome, T. Kanda, M. Ikeda. 2004. The toxicology and safety of apple polyphenol extract. Food Chem. Toxicol. 42: 959-967. [Medline]
  72. Shoji, T., S. Masumoto, N. Moriichi, H. Akiyama, T. Kanda, Y. Ohtake, Y. Goda. 2006. Apple procyanidin oligomers absorption in rats after oral administration: analysis of procyanidins in plasma using the porter method and high-performance liquid chromatography/tandem mass spectrometry. J. Agric. Food Chem. 54: 884-892. [Medline]



This article has been cited by other articles:


Home page
 J. Nutr.Home page
S. S. Percival
Grape Consumption Supports Immunity in Animals and Humans
J. Nutr., September 1, 2009; 139(9): 1801S - 1805S.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Holderness, J.
Right arrow Articles by Jutila, M. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Holderness, J.
Right arrow Articles by Jutila, M. A.
Right arrowPubmed/NCBI databases
*Substance via MeSH

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS