Compared to several other metal ions with similar chemical properties, zinc is relatively harmless. Only exposure to high doses has toxic effects, making acute zinc intoxication a rare event. In addition to acute intoxication, long-term, high-dose zinc supplementation interferes with the uptake of copper. Hence, many of its toxic effects are in fact due to copper deficiency. While systemic homeostasis and efficient regulatory mechanisms on the cellular level generally prevent the uptake of cytotoxic doses of exogenous zinc, endogenous zinc plays a significant role in cytotoxic events in single cells. Here, zinc influences apoptosis by acting on several molecular regulators of programmed cell death, including caspases and proteins from the Bcl and Bax families. One organ where zinc is prominently involved in cell death is the brain, and cytotoxicity in consequence of ischemia or trauma involves the accumulation of free zinc. Rather than being a toxic metal ion, zinc is an essential trace element. Whereas intoxication by excessive exposure is rare, zinc deficiency is widespread and has a detrimental impact on growth, neuronal development, and immunity, and in severe cases its consequences are lethal. Zinc deficiency caused by malnutrition and foods with low bioavailability, aging, certain diseases, or deregulated homeostasis is a far more common risk to human health than intoxication.
Zinc signals, i.e. a change of the intracellular concentration of free zinc ions in response to receptor stimulation, are involved in signal transduction in several immune cells. Here, the role of zinc signals in T-cell activation by IL-2 was investigated in the murine cytotoxic T-cell line CTLL-2 and in primary human T cells. Measurements with the fluorescent dyes FluoZin-3 and Zinquin showed that zinc is released from lysosomes into the cytosol in response to stimulation of the IL-2-receptor. Activation of the ERK-pathway was blocked by chelation of free zinc with N,N,N 0 ,N 0 -tetrakis-2(pyridyl-methyl)ethylenediamine, whereas zinc was not required for STAT5 phosphorylation. In addition, the key signaling molecules MEK and ERK were activated in response to elevated free intracellular zinc, induced by incubation with zinc and the ionophore pyrithione. Downstream of ERK activation, ERK-specific gene expression of c-fos and IL-2-induced proliferation was found to depend on zinc. Further experiments indicated that inhibition of MEK and ERK-dephosphorylating protein phosphatases is the molecular mechanism for the influence of zinc on this pathway. In conclusion, an increase of cytoplasmic free zinc is required for IL-2-induced ERK signaling and proliferation of T cells. Key words: IL-2 . T cells . Zinc Supporting Information available online IntroductionZinc signals have been observed in different cell types of the immune system, including monocytes, dendritic cells, and mast cells [1]. T-cell function is particularly susceptible to zinc deprivation, and zinc signals were suggested to activate protein kinase C in T cells [1,2]. Furthermore, zinc is involved in the activation of the Src-family kinase Lck by the TCR. Here, zinc ions are required for interactions at two protein/protein interface sites. First, they stabilize the interaction between Lck and CD4 or CD8, recruiting the kinase to the TCR signaling complex [3]. Second, zinc ions stabilize homodimerization of Lck, which promotes activating transphosphorylation between two Lck molecules [4]. Cellular zinc homeostasis is mediated by ten members of the ZnT family and 14 members of the Zrt-, Irt-like protein (ZIP) family of zinc transporters [5]. Intracellular localization for most of these transporters remains to be determined. So far, no nuclear zinc transporters were identified, even though there is evidence that nuclear and cytoplasmic zinc are differentially regulated [6]. In general, ZIP transport zinc into the cytoplasm, whereas ZnT transport zinc out of the cell or into cellular compartments, SHORT COMMUNICATION 1496including different vesicular structures [7]. Importantly, zinc accumulates in a lysosomal compartment of T cells, from which it is released by ZIP8 in response to TCR-mediated activation by antibodies against CD2, CD3, and CD28 [8].Previously, zinc was also shown to be required for T-cell activation by IL-2, a growth-factor that stimulates proliferation of T cells [9]. These data point to IL-2 signaling as another target for zinc in T cell...
Free zinc ions (Zn(2+)) participate in several signaling pathways. The aim of the present study was to investigate a potential involvement of Zn(2+) in the PI3K/Akt pathway of interleukin (IL)-2 signaling in T-cells. The IL-2 receptor triggers three major pathways, ERK1/2, JAK/STAT5, and PI3K/Akt. We have previously shown that an IL-2-mediated release of lysosomal Zn(2+) into the cytoplasm activates ERK1/2, but not STAT5. In the present study, Akt phosphorylation in response to IL-2 was abrogated by the Zn(2+) chelator N,N,N',N'-tetrakis-2(pyridyl-methyl)ethylenediamine, and was induced by treatment with Zn(2+) and the ionophore pyrithione. The latter were ineffective in cells that were treated with siRNA against the phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a phosphatase that degrades the lipid second messenger PI(3,4,5)P3, which is produced by PI3K and leads to activation of Akt. Inhibition of recombinant PTEN by Zn(2+)in vitro yielded an IC50 of 0.59 nM. Considering a resting free cytoplasmic Zn(2+) level of 0.2 nM in the T-cell line CTLL-2, this seems ideally suited for dynamic regulation by cellular Zn(2+). Oxidation with H2O2 and supplementation with Zn(2+) led to similar changes in the CD spectrum of PTEN. Moreover, Zn(2+) partially prevented the oxidation of cysteines 71 and 124. Hence, we hypothesize that zinc signals affect the IL-2-dependent PI3K/Akt pathway by inhibiting the negative regulator PTEN through binding with a sub-nanomolar affinity to cysteine residues that are essential for its catalytic activity.
The fetal tight junction molecule claudin 6 (CLDN6) is virtually absent from any normal tissue, whereas it is aberrantly and frequently expressed in various cancers of high medical need.We engineered 6PHU3, a T-cell-engaging bispecific single chain molecule (bi-(scFv)2) with anti-CD3/anti-CLDN6 specificities, and characterized its pharmacodynamic properties.Our data show that upon engagement by 6PHU3, T cells strongly upregulate cytotoxicity and activation markers, proliferate and acquire an effector phenotype. 6PHU3 exerts potent killing of cancer cells in vitro with EC50 values in the pg/mL range. Subcutaneous xenograft tumors in NSG mice engrafted with human PBMCs are eradicated by 6PHU3 treatment and survival of mice is significantly prolonged. Tumors of 6PHU3-treated mice are strongly infiltrated with activated CD4+, CD8+ T cells and TEM type cells but not Tregs and display a general activation of a mostly inflammatory phenotype.These effects are only observed upon bispecific but not monospecific engagement of 6PHU3. Together with the exceptionally cancer cell selective expression of the oncofetal tumor marker CLDN6, this provides a safeguard with regard to toxicity.In summary, our data shows that the concept of T-cell redirection combined with that of highly selective targeting of CLDN6-positive solid tumors is effective. Thus, exploring 6PHU3 for clinical therapy is warranted.
Mercury and lead are widespread in the environment, causing chronic exposure of a large population to low concentrations of these metals. While several studies demonstrated that low levels of both metals affect the immune system, little is known about underlying molecular mechanisms. The objective of this study was to investigate the impact of mercuric (Hg(2+)) and lead (Pb(2+)) ions on T cells. Up to 100 μM Pb(NO(3))(2) had no effect on cellular viability and proliferation. In contrast, HgCl(2) caused a concentration-dependent decline of viable leukocytes and especially of activated T cells. Additionally, Hg(2+) induced reactive oxygen species (ROS) generation accompanied by the loss of mitochondrial transmembrane potential, measured by Dihydrorhodamine and Rhodamine-123, respectively. The antioxidant N-acetylcysteine partially reversed the toxic effects of Hg(2+), pointing to an involvement of ROS. The major cytokine controlling T-cell survival and proliferation is interleukin (IL)-2. Hg(2+) had no effect on the secretion of IL-2, but on IL-2 mediated signal transduction pathways, reducing phosphorylation of the downstream kinases ERK1/2 and AKT. Moreover, Hg(2+) led to an arrest of the cells in the S phase of the cell cycle. Taken together, these data fit a model in which Hg(2+) disrupts mitochondria, and the resulting release of ROS inhibits IL-2-dependent signal transduction, reducing proliferation and survival of T cells.
As membrane proteins play an important role in a variety of life-threatening diseases, the development of therapeutic monoclonal antibodies against membrane proteins is of significant interest. Among many other requirements, the process of antibody drug development requires a set of tailor-made assays for the characterization of the antibodies and for monitoring their activity. Designing assays to characterize antibodies directed to membrane proteins is challenging, because the natural targets are often not available in a format that is compatible with a biochemical assay setup. Thus, alternatives that mimic the targeted membrane proteins are needed. In this study, we developed optimal peptidic mimotopes for the ELISA-based detection of the novel therapeutic antibody IMAB362 in biological samples. Initial hits were identified using phage display and these hits were optimized with the help of structure-activity relationship analysis on peptide microarrays. The optimized peptides showed binding constants in the low nanomolar to picomolar range, an improvement by a factor of up to 30 compared to the initial hits. The best mimotope (apparent KD = 0.15 nM) was successfully used for the ELISA-based quantification of IMAB362 in samples from a mouse pharmacokinetic study. The process described allows the rapid discovery of mimotopes for target proteins that are difficult to produce or handle, which can then be used in pre-clinical and clinical assays or for the purification of biological products.
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