Zinc (Zn) is an essential trace element with a particular relevance for the immune system. This is especially important for aging, because the age-related functional decline of the immune system (immunosenescence) and elevated unspecific inflammation in the elderly (inflammaging) are related to Zn deficiency, and have been shown to be partially reversible by Zn supplementation. To understand the impact of Zn on immunity on the molecular level, its functions in immune cells have been well investigated, including its role as a second messenger in T-cells and macrophages. There is sufficient evidence that different trace elements (TE) affect each other by sharing common binding sites in transport and regulatory proteins. Still, the interaction of Zn ions with other trace elements in the immune system remains insufficiently studied, so far. The present project aims to change this by addressing three experimental objectives. First, we will test the suitability of free serum Zn as an alternative marker for Zn status as part of the TE fingerprint. Its levels will be investigated with low molecular weight fluorescent probes in samples from the EPIC Potsdam subcohort (P1) and the TEhab cohort (P2). Due to limited sample volume availability, the existing method, which has been established by the applicant, will be adjusted to be applied to extremely low volumes. Second, free intracellular Zn concentrations and its distribution will be measured in cooperation with other projects. This includes fibroblasts (P6), C. elegans (P3), and murine cells and tissues (in cooperation with P4). Whereas techniques for measuring free Zn in isolated cells are readily available, the investigation in the intact worm will be established as part of the present project. Third, the impact of TE on two experimental model systems for Zn-responsive immune cell signaling and activation will be investigated in vitro. These are T-cells, especially the Zn-dependent activation of the Interleukin-2 signaling pathways, and macrophages, where Zn signals are a central regulator of Toll-like-receptor 4 signaling.