Therefore, we compared the effects of TPEN and Zn/TPEN on primary human T cells. Notably, Zn/TPEN had absolutely no impact on ERK1/2 phosphorylation, cellular survival, and proliferation, demonstrating that the effects of TPEN are mediated by zinc chelation (Supporting

Information Fig. 5). Our results demonstrate that zinc release from lysosomes into the cytoplasm plays a role in IL-2R IWR-1 datasheet signaling in T cells, in particular for ERK1/2 activation. It remains to be seen inasmuch other signaling pathways, e.g. other cytokine receptors, also trigger the release of zinc, because this can not be concluded from similarities in their signal transduction. It has previously been shown that the IL-1 receptor and TLR4, which share essentially the same signaling pathways, including ERK 35, differ significantly with regard to zinc signaling. TLR4 utilizes zinc signals, but none were observed in response to stimulation with IL-1 22. ERK activation in response to IL-2, which is essential for T-cell proliferation, depends on zinc. After stimulation of the IL-2-receptor, free zinc is released into the cytosol, where it inhibits MEK and ERK dephosphorylation. It remains unclear what triggers the zinc transporters to release zinc from zincosomes. The zinc

wave in mast cells depends on ERK activation 23, but we found a requirement Cabozantinib in vitro for zinc in ERK activation. Furthermore, unpublished results from our group indicate that inhibition of the MEK/ERK pathway does not affect IL-2-induced zinc signals. Here, the growing knowledge of zinc transporters and their regulation will certainly provide impulses in the future 36. Finally, the biological

significance of IL-2 is not only based on its role in T-cell proliferation, but also on its function in the development of regulatory T cells and T-cell memory formation 37. It remains to be seen to which degree zinc signals are involved in these major mechanisms of immune regulation. The murine cytotoxic T-cell line CTLL-2 was cultured at 37°C in a humidified 5% CO2 atmosphere. Cells were grown in RPMI 1640 (Lonza, Belgium) containing 10% heat-inactivated FBS (PAA, Germany), 2 mM L-glutamine, 100 U/mL penicillin, enough 100 μg/mL streptomycin, 1 mM sodium pyruvate (all from Lonza), 3.6 μL/L β-mercaptoethanol (Merck, Germany), and 30 U/mL recombinant human IL-2 (Peprotech, Germany). PBMC were isolated from heparinized peripheral venous blood from healthy donors by centrifugation over Ficoll-Hypaque (Biochrom, Germany) and cultured in RPMI 1640 containing 10% heat inactivated FBS, 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin. For enrichment of activated T lymphocytes, PBMC were incubated for 2 days with 2.5 μg/mL PHA. Monocytes and B cells were depleted by adherence to plastic and supernatants transferred for 4 days into fresh culture medium containing 50 U/mL IL-2, yielding T-cell populations that were > 95% CD3+ and > 93% CD25+.