Abstract
Purinergic signaling plays a complex role in inflammation. Nucleotides released by T lymphocytes, endothelial cells, and platelets during inflammation induce cellular responses by binding to receptors that regulate intracellular signaling pathways. Previous studies have found that purinergic signaling can have both proinflammatory and anti-inflammatory effects, but the roles of specific pathways in specific cell types are poorly understood. We investigated the role of the P2Y12 signaling pathway in the activation of T lymphocytes in vitro. We isolated peripheral blood mononuclear cells (PBMCs) from healthy donors and pretreated them with ADP (a P2Y12 agonist), AR-C69931MX (a P2Y12 antagonist), or both. We then stimulated PBMC using phytohemagglutinin (PHA) or anti-CD3/CD28 antibodies. We found that ADP affects T cell responses in term of cell activity and receptor expression through both P2Y12-dependent and P2Y12-independent pathways and other responses (cytokine secretion) primarily through P2Y12 -independent pathways. The ADP-mediated effect changed over time and was stimulus-specific.
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Abbreviations
- 2MeSADP:
-
2-methylthio-ADP
- GPCRs:
-
G protein-coupled receptors
- PBMCs:
-
Peripheral blood mononuclear cells
- PBS:
-
Phosphate-buffered saline
- PHA:
-
Phytohemagglutinin
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This work was supported by the American Heart Association grant 16SDG26980003 to EL.
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Supplemental Figure 1
Flow cytometric gating strategy to define T cell populations. Supplemental Fig. 2: CD4+, CD8+ and CD4+/CD25+ cell populations at time 0. The percentage of PBMC cell population was characterized at time 0. The percentage of CD4, CD8, CD4 + CD25, CD19 and CD14 (black dots) were analyzed in PBMC after isolation. The isotype control is shown in red. Data are expressed as % of expression ± S.E.M. (n = 3). Supplemental Fig. 3: Cell viability. Cell viability was determined with propidium iodide exclusion, while apoptosis was assessed as Annexin V expression. Cells negative for both were considered viable. (PPTX 205 kb)
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Vemulapalli, H., Albayati, S., Patwa, V.C. et al. ADP exerts P2Y12 -dependent and P2Y12 -independent effects on primary human T cell responses to stimulation. J. Cell Commun. Signal. 14, 111–126 (2020). https://doi.org/10.1007/s12079-019-00540-8
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DOI: https://doi.org/10.1007/s12079-019-00540-8