Response of gammadelta T Cells to plant-derived tannins

Abstract

Many pharmaceutical drugs arc isolated from plants used in traditional medicines, and new plant-derived pharmaceutical drugs continue to be identified. Relevant to this review, different plant-derived agonists for gammadelta T cells are described that impart effector functions upon distinct subsets of these cells. Recently, plant tannins have been defined as one class of gammadelta T cell agonist and appear to preferentially activate the mucosal population. Mucosal gammadelta T cells function to modulate tissue immune responses and induce epithelium repair. Select tannins, isolated from apple peel, rapidly induce immune gene transcription in gammadelta T cells, leading to cytokinc production and increased responsiveness to secondary signals. Activity of these tannin preparations tracks to the procyanidin fraction, with the procyanidin trimer (C1) having the most robust activity defined to date. The response to the procyanidins is evolutionarily conserved in that responses are seen with human, bovine, and murine gammadelta T cells, although human cells show less selectivity. Procyanidin-induced responses described in this review likely account for the expansion of mucosal gammadelta T cells seen in mice and rats fed soluble extracts of tannins. Use of procyanidins to activate gammadelta T cells may represent a novel approach for the treatment of tissue damage and autoimmune diseases.

Figure 1. Structures of common polyphenol subunits

A) Proposed structure for lignin, a complex, heterogeneous scaffold for carbohydrates. B) Pentagalloylglocose, the simplest hydrolysable tannin. The sugar core is oriented in the perpendicular plane. The five galloyl residues can be replaced, removed, or modified to increase diversity. C) Flavonoid families. D) Example of a tetrameric procyanidin, galloylated at the second procyanidin residue. Names of the monomeric form of each subunit are presented in parenthesis.

Figure 2. HPLC Fractionation of Procyanidins

HPLC fractionation of procyanidins from APP confirms the oligomeric nature of the active procyanidin fraction. The enriched oligomeric procyanidin preparation was fractionated by normal phase HPLC. A, Absorbance profile @ 280nm for the initial, procyanidin fractionation and the biologically active fraction, #24. B, Expression of bovine IL2Rα was measured in the presence of fractions obtained from the HPLC separation. Data represent the lowest dilution of each fraction in medium required to achieve 30% activation. These results are representative of activity from two HPLC fractionations.

Figure 3. Purified Procyanidin Dimers and Trimer Induce Human γδ T Cell Proliferation in response to IL-15

Human PBMCs from 3 donors were CFSE-labeled and cultured with IL-15 (1 ng/ml) and either APP, catechin (Axxora, San Diego, CA), epicatechin (Sigma, St. Louis, MO), procyanidin B1 (PB1; Extrasynthese, Genay France), procyanidin B2 (PB2; Extrasynthese, Genay France), PC1 (Phytolab, Vestenbergsgreuth, Germany), or medium only. Dilutions of procyanidins or APP inducing peak responses were selected and compared for their γδ T cell proliferative ability. Data represent the average fold increase of γδ T cell proliferation in the procyanidin-treated samples versus the medium control (1 ng/ml IL-15) wells. Error bars represent the standard error of the mean. P-values for differences in means are *, P < 0.05; ***, P < 0.001.

Figure 4. At non-toxic concentrations, PC1 is a better Vδ1 agonist than APP

Human PBMC cultures were maintained for 48h with various concentrations of PC1 or APP under the conditions previously described. After the culture period, cells were stained with mAbs to Vδ1 TCR, Vδ2 TCR, and CD69 then analyzed by flow cytometry. Cultures not inducing toxicity were analyzed. Toxic cultures were determined by flow cytometry light scatter (FSC/SSC). Cultures with a ratio of healthy lymphocytes to cellular debris less than medium controls were eliminated. Maximum concentrations for baseline cell survival were 31.6 µg/mL and 5.6 µg/mL for PC1 and APP, respectively. Values represent the average fold increase in CD69⁺ cells compared to the medium controls for four donors. Error bars represent SEM.

Figure 5. γδ T cell Activation Cannot be Explained by Procyanidin-induced Cell Death

Semi-toxic concentrations of PC1, but not its monomeric subunit, induce γδ T cell proliferation. CFSE-labeled human PBMCs were cultured with IL-15 (1 ng/ml) and medium alone or various concentrations of purified epicatechin or purified PC1 for 5 days. Cultures were analyzed for lymphocyte survival by light scatter (FACS) where the maximum survival was medium only. Cell cultures between 30–60% lymphocyte death were analyzed for γδ T cell proliferation. Bars represent the AVG γδ T cell proliferation and SD from each of the 3 cultures found to induce this range of cell death in culture.