Ca2+ signals are essential for T-cell proliferation, while Zn2+ signals are necessary for T helper cell 1 differentiation.

The regulation of T-cell fate is crucial for the balance between infection control and tolerance. Calcium (Ca2+) and zinc (Zn2+) signals are both induced after T-cell stimulation, but their specific roles in the fate of activation and differentiation remain to be elucidated. Are Zn2+- and Ca2+ signals responsible for different aspects in T-cell activation and differentiation and do they act in concert or in opposition? It is crucial to understand the interplay of the intracellular signals to influence the fate of T cells in diseases with undesirable T-cell activities or in Zn2+-deficient patients. Human peripheral blood mononuclear cells were stimulated with the Zn2+ ionophore pyrithione and thapsigargin, an inhibitor of the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA). Intracellular Zn2+ and Ca2+ signals were monitored by flow cytometry and ELISA, quantitative PCR and western blot were used to evaluate T-cell differentiation and the underlying molecular mechanism. We found that Zn2+ signals upregulated the early T-cell activation marker CD69, interferon regulatory factor 1 (IRF-1), and Krüppel-like factor 10 (KLF-10) expression, which are important for T helper cell (Th) 1 differentiation. Ca2+ signals, on the other hand, increased T-bet and Forkhead box P3 (FoxP3) expression and interleukin (IL)-2 release. Most interestingly, the combination of Zn2+ and Ca2+ signals was indispensable to induce interferon (IFN)-γ expression and increased the surface expression of CD69 by several-fold. These results highlight the importance of the parallel occurrence of Ca2+ and Zn2+ signals. Both signals act in concert and are required for the differentiation into Th1 cells, for the stabilization of regulatory T cells, and induces T-cell activation by several-fold. This provides further insight into the impaired immune functions of patients with zinc deficiency.

Zinc Ionophore Pyrithione Mimics CD28 Costimulatory Signal in CD3 Activated T Cells.

Zinc is an essential trace element that plays a crucial role in T cell immunity. During T cell activation, zinc is not only structurally important, but zinc signals can also act as a second messenger. This research investigates zinc signals in T cell activation and their function in T helper cell 1 differentiation. For this purpose, peripheral blood mononuclear cells were activated via the T cell receptor-CD3 complex, and via CD28 as a costimulatory signal. Fast and long-term changes in intracellular zinc and calcium were monitored by flow cytometry. Further, interferon (IFN)-γ was analyzed to investigate the differentiation into T helper 1 cells. We show that fast zinc fluxes are induced via CD3. Also, the intracellular zinc concentration dramatically increases 72 h after anti-CD3 and anti-CD28 stimulation, which goes along with the high release of IFN-γ. Interestingly, we found that zinc signals can function as a costimulatory signal for T helper cell 1 differentiation when T cells are activated only via CD3. These results demonstrate the importance of zinc signaling alongside calcium signaling in T cell differentiation.

Leukemia cells accumulate zinc for oncofusion protein stabilization.

Acute promyelocytic leukemia (APL) and chronic myeloid leukemia (CML) are both hematological malignancies characterized by genetic alterations leading to the formation of oncofusion proteins. The classical chromosomal aberrations in APL and CML result in the PML-RARα and BCR-ABL1 oncofusion proteins, respectively. Interestingly, our flow cytometric analyses revealed elevated free intracellular zinc levels in various leukemia cells, which may play a role in stabilizing oncofusion proteins in leukemia and thus support cell proliferation and malignancy. Long-term zinc deficiency resulted in the degradation of PML-RARα in NB4 cells (APL cell line) and of BCR-ABL1 in K562 cells (CML cell line). This degradation may be explained by increased caspase 3 activity observed in zinc deficient cells, whereas zinc reconstitution normalized the caspase 3 activity and abolished zinc deficiency-induced oncofusion protein degradation. In NB4 cells, fluorescence microscopic images further indicated enlarged and enriched lysosomes during zinc deficiency, suggesting increased rates of autophagy. Moreover, NB4 cells exhibited increased expression of the zinc transporters ZIP2, ZIP10 and ZnT3 during zinc deficiency and revealed excessive accumulation of zinc in contrast to healthy peripheral blood mononuclear cells (PBMCs), when zinc was abundantly available extracellularly. Our results highlight the importance of altered zinc homeostasis for some characteristics in leukemia cells, uncover potential pathways underlying the effects of zinc deficiency in leukemia cells, and provide potential alternative strategies by which oncofusion proteins can be degraded.

The Influence of Zinc and Citrate on T Cell Activation and Differentiation in Mixed Lymphocyte Cultures.

SCOPE: Zinc is important for a balanced immune system, but the mechanisms are not yet fully elucidated. One possibility is an interaction of zinc with the tricarboxylic acid cycle (TCA), in which zinc inhibits the mitochondrial aconitase leading to an increase in intracellular citrate concentration as described for prostate cells. Therefore, the immune modulatory effects of zinc and citrate and their interaction in mixed lymphocyte cultures (MLC) are studied. METHODS AND RESULTS: After allogeneic (MLC) or superantigen stimulation, the interferon-γ (IFNγ) production is quantified by ELISA and T cell subpopulations are determined by Western Blot. Intracellular concentrations of citrate and zinc are measured. Zinc and citrate reduce the IFNγ expression and the pro-inflammatory T helper cells (Th) 1 and Th17 in MLC. While zinc increases regulatory T cells, citrate reduces them. After superantigen stimulation IFNγ production is decreased only by citrate but increased by zinc. Zinc does not affect citrate concentration, while citrate impairs zinc uptake. Thus, zinc and citrate independently regulate IFNy expression. CONCLUSION: These results may explain the immunosuppressive effect of blood products anticoagulated by citrate. In addition, high citrate consumption may lead to immunosuppressive effects, so upper limits for citrate should be established.

Zinc stabilized Nrf2 by inhibition of HDAC3 in human peripheral blood mononuclear cells.

BACKGROUND: The transcription factor Nuclear factor erythroid 2-related factor 2 (Nrf2) induces several detoxifying proteins, which also include NAD(P)H quinone dehydrogenase 1 (NQO1) and heme oxygenase 1 (HO-1). The expression of these Nrf2-regulated proteins is important for the maintenance of the redox homeostasis in cells. The aim of this study was to investigate the effect of tert-butyl-hydrochinone (tBHQ) stimulation on human PBMC under normal condition and zinc depletion, respectively. METHOD: Human peripheral blood mononuclear cells (PBMC) were treated with the Nrf2 activator tBHQ in combination with zinc to examine a possible correlation between zinc and redox homeostasis. Therefore, mRNA expression of Nrf2 and its downstream molecules NQO1 and HO-1 were investigated, as well as the protein synthesis of these. In addition, the effect of zinc on histone deacetylase 3 (HDAC3), which is a negative regulator for Nrf2 activity, was analyzed. RESULTS: Either mRNA, protein expression or both of Nrf2, NQO1 and HO-1 are influenced by zinc. The analysis of HDAC3 shows a negative correlation between its activity and increasing zinc concentrations. By inhibiting HDAC3 zinc stabilizes Nrf2. CONCLUSION: The results indicate that zinc emphasizes the induction of Nrf2 by its activator tBHQ through increasing gene and protein expression. Additionally, zinc supplementation inhibits HDAC3 activity resulting in reduced Keap1 mRNA expression and thereby stabilizing cytoplasmatic Nrf2. These findings suggests that zinc supplementation has beneficial effects on the redox balance in human cells.

Proton-Pump Inhibitors Suppress T Cell Response by Shifting Intracellular Zinc Distribution.

Proton-pump inhibitors (PPI), e.g., omeprazole or pantoprazole, are the most widely used drugs for various gastrointestinal diseases. However, more and more side effects, especially an increased risk of infections, have been reported in recent years. The underlying mechanism has still not yet been fully uncovered. Hence, in this study, we analyzed the T cell response after treatment with pantoprazole in vitro. Pantoprazole preincubation reduced the production and secretion of interferon (IFN)-γ and interleukin (IL)-2 after the T cells were activated with phytohemagglutinin (PHA)-L or toxic shock syndrome toxin-1 (TSST-1). Moreover, a lower zinc concentration in the cytoplasm and a higher concentration in the lysosomes were observed in the pantoprazole-treated group compared to the untreated group. We also tested the expression of the zinc transporter Zrt- and Irt-like protein (Zip)8, which is located in the lysosomal membrane and plays a key role in regulating intracellular zinc distribution after T cell activation. Pantoprazole reduced the expression of Zip8. Furthermore, we measured the expression of cAMP-responsive element modulator (CREM) α, which directly suppresses the expression of IL-2, and the expression of the phosphorylated cAMP response element-binding protein (pCREB), which can promote the expression of IFN-γ. The expression of CREMα was dramatically increased, and different isoforms appeared, whereas the expression of pCREB was downregulated after the T cells were treated with pantoprazole. In conclusion, pantoprazole downregulates IFN-γ and IL-2 expression by regulating the expression of Zip8 and pCREB or CREMα, respectively.