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. 2018 Dec 11:9:2949.
doi: 10.3389/fimmu.2018.02949. eCollection 2018.

Human T2R38 Bitter Taste Receptor Expression in Resting and Activated Lymphocytes

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Human T2R38 Bitter Taste Receptor Expression in Resting and Activated Lymphocytes

Hoai T T Tran et al. Front Immunol. .

Abstract

The human G-protein-coupled bitter taste receptor T2R38 has recently been demonstrated to be expressed on peripheral blood neutrophils, monocytes and lymphocytes. To further define a potential contribution of the T2R38 receptor in adaptive immune response, the objective of this study was to analyze its expression in resting and activated lymphocytes and T cell subpopulations. Freshly isolated PBMC from healthy donors were used for expression analysis by flow cytometry. Quantum™ MESF beads were applied for quantification in absolute fluorescence units. Activation methods of T cells were anti-CD3/CD28, phytohaemagglutinin (PHA) or phorbol 12-myristate 13-acetate (PMA) together with ionomycin. Lymphocytes from young donors expressed higher levels of T2R38 compared to the elderly. CD3+ T cells expressed higher levels that CD19+ B cells. Receptor expression followed T cell activation with an upregulation within 24 h and a peak at 72 h. Higher levels of T2R38 were produced in lymphocytes by stimulation with anti-CD3/CD28 compared to PHA or PMA/ionomycin. Both subpopulations of CD4+ as well as CD8+ T cells were found to express the T2R38 receptor; this was higher in CD4+ than CD8+ cells; the amount of T2R38 in central and effector memory cells was higher as compared to naïve cells, although this was not statistically significant for CD8+ cells without prior activation by anti-CD3/CD28. Upon treatment of PBMC with the natural T2R38 agonist goitrin Calcium flux was activated in the lymphocyte population with functional T2R38 receptor at >20 μM which was completely blocked by phospholipase Cβ-2 inhibitor U73211. Further, goitrin selectively inhibited TNF-alpha secretion in PBMC with functional T2R38. This quantitative analysis of T2R38 expression in distinct PBMC subsets may provide a basis for understanding the significance of bitter compounds in immune modulation. Whether these findings can have implications for the treatment of inflammatory and immunologic disorders by bitter tasting pharmaceuticals or foods needs further investigation.

Keywords: G protein coupled receptor (GPCR); T2R38; aging; hTAS2R38 gene; human T cells; human bitter taste receptor (T2R).

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Figures

Figure 1
Figure 1
Expression of T2R38 receptor in human PBMC. (A) Age-related T2R38 expression in PBMC from young (20–35 years, n ≥ 10) and elderly (60–90 years, n = 9) subjects. A representative staining from one person is shown for each cell type; T2R38 (continuous line) and rabbit IgG (dotted line). (B) T2R38 expression in CD3+ T cells from young (n = 7) and elderly (n = 7) donors (C) Sex difference of T2R38 expression in lymphocytes from male and female donors (n = 10). (D) T2R38 expression in CD3-PE stained T cells and CD19-APC stained B cells from human volunteers, (n = 6). A representative staining from one person is shown; CD3+ (continuous line) and CD19+ (dotted line). (E) Haplotype analysis was done using amplification of a TAS2R38 864bp fragment followed by sequencing. PAV: proline, alanine and valine; AVI: alanine, valine and isoleucine (n = 19). T2R38 expression (delta MESF) was quantified by Quantum Alexa Fluor 488 MESF using cytofluorometry (Supplementary Figure S1A), IgG was used as isotype control. Each dot presents one donor in the graphs. The gating strategy is shown in Supplementary Figure S1B. Significance of difference was calculated relatively to the respective control, *p < 0.05, **p < 0.01.
Figure 2
Figure 2
Effect of activation on T2R38 expression in PBMC. (A) PBMC were stimulated with different concentrations of PMA/ionomycin (1 μg/ml), PHA or anti-CD3/CD28 mAbs for 72 h (n ≥ 5) (B) Isolated PBMC from young and elderly individuals were stimulated with 1 ng/ml CD3/CD28 for 72 h (n ≥ 5) (C) PBMC were stimulated with 1 ng/ml CD3/CD28 for the indicated time points, activated cells were determined by the percentage of blast cells (n ≥ 3) (D) PBMC were stimulated with 1 ng/ml CD3/CD28 and T2R38 expression on CD69+/CD25+ CD3+T lymphocytes determined at the indicated time points (n = 5). A representative staining of surface markers CD69+/CD25+ at day 2 from one subject is shown as scattergram. T2R38 expression (delta MESF) was assessed by Quantum Alexa Fluor 488 MESF beads in comparison to rabbit IgG isotype control; bars are means + SD (A,B) or means ± SD (C,D). Significance of difference was calculated relatively to the respective control, *p < 0.05; **p < 0.01. SC, solvent control. The gating strategy is shown in Supplementary Figure S2.
Figure 3
Figure 3
T2R38 expression status in T cell subsets. T2R38 expression was quantified in (A,B) freshly isolated cells (n ≥ 6) or (C,D) CD3/CD28-stimulated cells after 72 h (n ≥ 5). Each dot represents one donor, bars are means + SD. NA, naïve, CM, central memory, EM, effector memory cells. Representative histograms of different stains from one donor are shown. The gating strategy is given in Supplementary Figure S3. T2R38 expression (delta MESF) was assessed by Quantum Alexa Fluor 488 MESF beads in comparison to rabbit IgG isotype control. Significance of difference was calculated relatively to the respective control, *p < 0.05, **p < 0.01.
Figure 4
Figure 4
Analysis of calcium flux and TNF-alpha secretion. (A) FACS-based measurement of calcium flux was detected in lymphocytes (PAV haplotype) probed with Fluor-4 AM (FEM:516nm) after treatment with different concentrations of goitrin alone or 1 h pre-incubation with 10 μM PLC-β2 inhibitor U73122. Baseline fluorescence was recorded for 60 s before addition of goitrin and fluorescence was measured for a total of 800 s. Calcium response was calculated as the ratio of the maximum peak post stimulation to basal level using FlowJo software. The gating strategy is shown in Supplementary Figure S4. Bars are means + SD, n ≥ 8. Significance of difference was calculated relatively to the respective control, *p < 0.05. (B) PBMC with functional (PAV haplotype) and non-functional (AVI/AVI) T2R38 receptor were exposed to 100 μM goitrin or 0.01% DMSO for 72 h w/wo IL-2 and TNF-alpha secretion was analyzed using an ELISA kit. Results are mean values + SD, **p < 0.01. Significance of the difference was determined compared to the respective solvent control (SC, 0.01% DMSO).

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