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...

The trace element zinc is a crucial cofactor for many proteins involved in cellular processes like differentiation, proliferation and apoptosis. Zinc homeostasis is tightly regulated and disturbance of this homeostasis due to genetic defects, zinc deficiency, or supplementation influences the development and the progression of various infectious and autoimmune diseases. The immune system is strongly impaired during zinc deficiency, predominantly the cell-mediated response by T-lymphocytes. During zinc deprivation T-lymphocyte development, polarization into effector cells, and function are impaired. This leads to reduced T-cell numbers, a decreased ratio of type 1 to type 2 T-helper cells with reduced production of T-helper type 1 cytokines like interferon-gamma, and compromised T-cell mediated immune defense. Accordingly, disturbed zinc homeostasis increases the risk for infections, and zinc supplementation restores normal immune function. Furthermore, several disorders, like mycobacterial infections, asthma, diabetes, and rheumatoid arthritis are accompanied by decreased zinc levels and in some cases disease progression can be affected by zinc supplementation. On the molecular level, apoptosis of T-cell precursors is influenced by zinc via the Bcl-2/Bax ratio, and zinc ions inhibit caspases-3, -6, -7, and -8. In mature T-cells, zinc interacts with kinases involved in T-cell activation, like protein kinase C and the lymphocyte protein tyrosine kinase (Lck), while higher zinc concentrations are inhibitory, reducing the activities of the interleukin-1 receptor-associated kinase (IRAK) and calcineurin. Taken together, zinc homeostasis influences T-lymphocytes via several molecular targets, leading to a modulation of T-cell-dependent immune responses.

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It was reported previously that zinc-deficient mice show impaired lymphopoiesis. At the same time, monocyte numbers in these animals are increased, indicating a negative impact of zinc on monocyte development. Here, we investigate the role of zinc homeostasis in the differentiation of myeloid precursors into monocytes. Reduced gene expression of several zinc transporters, predominantly from the Zip family, was observed during 1 alpha, 25-dihydroxyvitamin D(3) (1,25D(3))-induced differentiation of HL-60 cells. This was accompanied by a reduction of intracellular-free zinc, measured by FluoZin-3. Amplifying this reduction with the zinc chelator TPEN or zinc-depleted cell-culture medium enhanced 1,25D(3)-induced expression of monocytic surface markers CD11b and CD14 on HL-60, THP-1, and NB4 cells. In contrast, differentiation of NB4 cells to granulocytes was not zinc-sensitive, pointing toward a specific effect of zinc on monocyte differentiation. Further, monocyte functions, such as TNF-alpha secretion, phagocytosis, and oxidative burst, were also augmented by differentiation in the presence of TPEN. The second messenger cAMP promotes monocyte differentiation. We could show that zinc inhibits the cAMP-synthesizing enzyme adenylate cyclase, and chelation of zinc by TPEN increases cAMP generation after stimulation with the adenylate cyclase activator forskolin. Based on our in vitro results and the in vivo observations from the literature, we suggest a model in which the intracellular-free zinc concentration limits AC activity, and the decrease of zinc after 1,25D(3) treatment promotes differentiation by relieving AC inhibition. Thus, cellular zinc homeostasis acts as an endogenous modulator of monocyte differentiation.

The nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 (NF-κB1) is a master regulator of immune and inflammatory responses.1,2 NF-κB1 belongs to the NF-κB/Rel family of transcription factors that consists of five members in humans: NF-κB1 (p105/p50), NF-κB2 (p100/p52), RelA, c-Rel, and RelB. The p105 and p100 precursors are proteolytically processed by the proteasome to generate the shorter p50 and p52 isoforms. Homo-and heterodimers are formed by p50, p52 and the Rel proteins. Unstimulated, these dimeric complexes are sequestered in the cytoplasm by inhibitory IκB proteins in an inactive state. Upon stimulation, the phosphorylation and subsequent degradation of IκB proteins is rapidly triggered, releasing the NF-κB/Rel complexes to enter the nucleus where they bind to DNA at κB sites and activate or repress the expression of their target genes. 3 Recently, heterozygous mutations affecting the NFKB1 gene were identified in three human families. Haploinsufficiency of NF-κB1 was causative for combined variable immunodeficiency (CVID) characterized by recurrent infections due to immunoglobulin deficiency (pan-IgG, IgA and/or IgM). 4 We report here on two pediatric patients from unrelated families with two novel NFKB1 gene mutations identified by whole-exome sequencing ( Figure 1A,B). Both patients had early onset of disease during their teenage years and presented in addition to hypogammaglobulinemia or selective IgA deficiency with a striking lack of specific antibodies (clinical characteristics are summarized in Table 1 and Online Supplementary Table S1).Patient 1 is a now 26-year old female who first presented with recurrent autoimmune hemolytic anemia at the age of 14. Hypogammaglobulinemia (IgG2 subclass deficiency), deficient production of specific antibodies, decreased class-switched and memory B cells, naïve CD4-positive and regulatory T cells, increased activated and double-negative T cells (DNT cells, CD4 -CD8 -TCRα/β + ), autoimmune phenomena (hemolytic anemia, thrombocytopenia and leukopenia), lymphadenopathy, and hepatosplenomegaly were observed. She developed chronic lung disease with bronchiectasis, frequent respiratory tract infections and pneumonia. Infections with viral, bacterial and fungal pathogens were frequent. She suffered from intractable abdominal pain and bloody diarrhea without evidence of infection. After a liver biopsy she developed pancolitis with subsequent sepsis and multi-organ failure and was successfully resuscitated. The patient is being treated with intravenous immunoglobulin. Steroids were intermittently given to reduce pulmonary infiltrates with partial response. Mycophenolate mofetil stabilized blood counts, but pulmonary symptoms and infections remained.To identify the genetic cause of disease whole exome sequencing was performed for the patient and her family (Online Supplementary Methods, Online Supplementary Table S2). A heterozygous de novo NFKB1 frameshift mutation was detected ( Figure 1A). The NFKB1 gene encodes two proteins: p50 and its precursor ...

Serine/threonine kinase 4 (STK4) deficiency is an autosomal recessive genetic condition that leads to primary immunodeficiency (PID) typically characterized by lymphopenia, recurrent infections and Epstein Barr Virus (EBV) induced lymphoproliferation and -lymphoma. State-of-the-art treatment regimens consist of prevention or treatment of infections, immunoglobulin substitution (IVIG) and restoration of the immune system by hematopoietic stem cell transplantation. Here, we report on two patients from two consanguineous families of Turkish (patient P1) and Moroccan (patient P2) decent, with PID due to homozygous STK4 mutations. P1 harbored a previously reported frameshift (c.1103 delT, p.M368RfsX2) and P2 a novel splice donor site mutation (P2; c.525+2 T>G). Both patients presented in childhood with recurrent infections, CD4 lymphopenia and dysregulated immunoglobulin levels. Patient P1 developed a highly malignant B cell lymphoma at the age of 10 years and a second, independent Hodgkin lymphoma 5 years later. To our knowledge she is the first STK4 deficient case reported who developed lymphoma in the absence of detectable EBV or other common viruses. Lymphoma development may be due to the lacking tumor suppressive function of STK4 or the perturbed immune surveillance due to the lack of CD4+ T cells. Our data should raise physicians' awareness of [1] lymphoma proneness of STK4 deficient patients even in the absence of EBV infection and [2] possibly underlying STK4 deficiency in pediatric patients with a history of recurrent infections, CD4 lymphopenia and lymphoma and unknown genetic make-up. Patient P2 experienced recurrent otitis in childhood, but when she presented at the age of 14, she showed clinical and immunological characteristics similar to patients suffering from Autoimmune Lymphoproliferative Syndrome (ALPS): elevated DNT cell number, non-malignant lymphadenopathy and hepatosplenomegaly, hematolytic anemia, hypergammaglobulinemia. Also patient P1 presented with ALPS-like features (lymphadenopathy, elevated DNT cell number and increased Vitamin B12 levels) and both were initially clinically diagnosed as ALPS-like. Closer examination of P2, however, revealed active EBV infection and genetic testing identified a novel STK4 mutation. None of the patients harbored typically ALPS-associated mutations of the Fas receptor mediated apoptotic pathway and Fas-mediated apoptosis was not affected. The presented case reports extend the clinical spectrum of STK4 deficiency.

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