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Nuclear factor 'κ‐light‐chain‐enhancer' of activated B cells (NF‐κB) signaling is a signaling pathway used by most immune cells to promote immunostimulatory functions. Recent studies have indicated that regulatory T cells (Treg) differentially integrate TCR‐derived signals, thereby maintaining their suppressive features. However, the role of NF‐κB signaling in the activation of human peripheral blood (PB) Treg has not been fully elucidated so far. We show that the activity of the master transcription factor forkhead box protein 3 (FOXP3) attenuates p65 phosphorylation and nuclear translocation of the NF‐κB proteins p50, p65, and c‐Rel following activation in human Treg. Using pharmacological and genetic inhibition of canonical NF‐κB signaling in FOXP3‐transgenic T cells and PB Treg from healthy donors as well as Treg from a patient with a primary NFKB1 haploinsufficiency, we validate that Treg activation and suppressive capacity is independent of NF‐κB signaling. Additionally, repression of residual NF‐κB signaling in Treg further enhances interleukin‐10 (IL‐10) production. Blockade of NF‐κB signaling can be exploited for the generation of in vitro induced Treg (iTreg) with enhanced suppressive capacity and functional stability. In this respect, dual blockade of mammalian target of rapamycin (mTOR) and NF‐κB signaling was accompanied by enhanced expression of the transcription factors FOXP1 and FOXP3 and demethylation of the Treg‐specific demethylated region compared to iTreg generated under mTOR blockade alone. Thus, we provide first insights into the role of NF‐κB signaling in human Treg. These findings could lead to strategies for the selective manipulation of Treg and the generation of improved iTreg for cellular therapy. [ABSTRACT FROM AUTHOR]

The organometallic AuI bis‐N‐heterocyclic carbene complex [Au(9‐methylcaffeine‐8‐ylidene)2]+ (AuTMX2) was previously shown to selectively and potently stabilise telomeric DNA G‐quadruplex (G4) structures. This study sheds light on the molecular reactivity and mode of action of AuTMX2 in the cellular context using mass spectrometry‐based methods, including shotgun proteomics in A2780 ovarian cancer cells. In contrast to other metal‐based anticancer agents, this organogold compound is less prone to form coordinative bonds with biological nucleophiles and is expected to exert its drug effects mainly by non‐covalent interactions. Global protein expression changes of treated cancer cells revealed a multimodal mode of action of AuTMX2 by alterations in the nucleolus, telomeres, actin stress‐fibres and stress‐responses, which were further supported by pharmacological assays, fluorescence microscopy and cellular accumulation experiments. Proteomic data are available via ProteomeXchange with identifier PXD020560. [ABSTRACT FROM AUTHOR]

Background: Regulatory T lymphocytes (Treg) play an important role in preventing allergic diseases. We characterized Treg expansion kinetics, marker profiles, and recirculation behavior in allergen‐challenged mice, which had been pretreated with IL‐2/αIL‐2 complexes in the presence or absence of allergen. Moreover, the ability of induced Treg to control airway hyperreactivity and effector functions of lung T cells was determined. Methods: Humanized TCR/HLA‐transgenic allergy mice were treated in vivo with recombinant IL‐2 complexed to the anti‐IL‐2 mAb JES6‐1 in the presence or absence of mugwort pollen extract (MPE) on days 0‐2. Afterward, they were intranasally challenged with MPE (days 13‐15) followed by determination of airway hyperreactivity and lung T cell effector functions. Multiparametric flow cytometry on peripheral blood T cells was performed on a daily basis. Results: IL‐2/αIL‐2 complexes highly efficiently expanded peripheral Treg cells, while concomitant allergen exposure altered the phenotype of expanded Treg cells. Notably, application of allergen together with IL‐2/αIL‐2 complexes induced expression of Treg marker molecules CTLA4, NRP1, Helios, and GITR on conventional T cells. Apart from CD25, GARP was identified as the most reliable surface‐expressed lineage discrimination marker of Treg expanded in the presence of IL‐2/αIL‐2 complexes and allergen. Finally, IL‐2/αIL‐2 complex‐expanded Treg cells could be recalled upon allergen challenge, which was associated with suppression of lung‐specific Th2 responses long after initial treatment. Conclusion: The characterization of reliable surface and transcription markers of IL‐2/αIL‐2 complex‐expanded Treg along with their expansion kinetics and function will help to identify protocols for their long‐term expansion in vivo. [ABSTRACT FROM AUTHOR]

The signal transducer and activator of transcription (STAT) proteins are important mediators for the integration of extrinsic signals provided by cytokines and hormones and thereby adapt cellular processes to their surroundings. In the past decade, the involvement of STAT3 in the regulation of T-cell responses has become a topic of increasing interest. STAT3 is activated in response to multiple cytokines, many of which have been shown to influence T-cell responses. Interestingly, many of these factors have been described with apparent opposing roles, such as the highly pro-inflammatory potency of IL-6 and the anti-inflammatory properties of IL-10, thus raising the possibility that STAT3 signaling may fulfill diverse roles in CD4+ T-cells. Here, we review the contribution of STAT3 to the induction and function of both peripherally induced as well as thymus-derived regulatory T-cells. Indeed, experimental approaches as well as studies of human patients suffering from e.g. Job's (hyper IgE) syndrome or inflammatory bowel disease (IBD) have now established a clear-cut role for the IL-10/STAT3 axis in immune tolerance; further understanding of these processes could lead to novel therapeutic approaches for autoimmune diseases. [ABSTRACT FROM AUTHOR]

Background Neutropaenic patients are at a high risk of contracting severe infections. In particular, in these patients, parameters with a high negative predictive value are desirable for excluding infection or bacteraemia. This study evaluated sepsis biomarkers in neutropaenic patients suffering from systemic inflammatory response syndrome ( SIRS). Further, the predictive capacities of evaluated biomarkers in neutropaenic SIRS patients were compared to non-neutropaenic SIRS patients. Material and methods In this prospective observational cohort study, patients with clinically suspected sepsis were screened. The predictive capacities of procalcitonin ( PCT), C-reactive protein and lipopolysaccharide-binding protein ( LBP) in neutropaenic SIRS patients were evaluated in terms of their potential to identify infection or bacteraemia and were compared to results for non-neutropaenic SIRS patients. To select an appropriate control cohort, propensity score matching was applied, balancing confounding factors between neutropaenic and non-neutropaenic SIRS patients. Results Of 3370 prospectively screened patients with suspected infection, 51 patients suffered from neutropaenic SIRS. For the identification of infection, none of the assessed biomarkers presented a clinically relevant discriminatory potency. Lipopolysaccharide-binding protein and PCT demonstrated discriminatory capacity to discriminate between nonbacteraemic and bacteraemic SIRS in patients with neutropaenia [receiver-operating characteristics-area under the curves ( ROC- AUCs): 0·860, 0·818]. In neutropaenic SIRS patients, LBP had a significantly better ROC- AUC than in a comparable non-neutropaenic patient cohort for identifying bacteraemia ( P = 0·01). Conclusion In neutropaenic SIRS patients, none of the evaluated biomarkers was able to adequately identify infection. LBP and PCT presented a good performance in identifying bacteraemia. Therefore, these markers could be used for screening purposes to increase the pretest probability of blood culture analysis. [ABSTRACT FROM AUTHOR]

Signal transducer and activator of transcription 3 (STAT3) integrates key signals of cell surface immune receptors, yet its precise role in cluster of differentiation (CD)4+ T cells is not well-established. Current research has indicated T-helper cell 17-inducing roles but also tolerogenic roles. To address this issue, human T cells were transduced with the constitutively active STAT3 mutant STAT3C. Following stimulation, STAT3C+ T cells up-regulated IUO (4.1 ± 0.5-fold; P< 0.001) and granzyme B (2.5 ± 1.2, P< 0.05) secretion, combined with significantly reduced IFN-g (35 ± 5%), IL-2 (57 ± 4%), TNF-α (64 ± 8%), and IL-13 (89 ± 3%) secretion (P< 0.001). CD3/CD2-or CD3/CD28-activated STAT3C+ T cells revealed reduced proliferation (53.4 ± 23.5% and 70.5 ± 10.4%, respectively), which was independent of IUO production and significantly suppressed effector T cell proliferation by 68.7 6 10.6% and 65.9 6 2.6%, respectively (P< 0.001). Phenotypically, STAT3C-transgenic CD4+ T cells resembled effector T cells regarding expression of T regulatory cell markers, but up-regulated granzyme B expression levels by 2.4-fold (P< 0.05). Suppression was cell contact dependent and mediated by granzyme B-induced cell death, but was independent of IL- 10 and TGF-β. Notably, peripheral blood CD4+CD45RA-lymphocyte activation gene-3+CD49+ type 1 regulatory T cells revealed activation-induced hyperphosphorylation of STAT3. In agreement, pharmacological inhibition of STAT3 activation partially reverted hyporesponsiveness of peripheral type 1 regulatory T cells (increasing their division index from 0.46 6 0.11 to 0.89 6 0.04; P< 0.01). These observations indicate a clear-cut relation between activation of STAT3 and the acquisition of a tolerogenic program, which is also used by peripheral blood type 1 regulatory T cells. [ABSTRACT FROM AUTHOR]

Naturally occurring CD4+CD25high fork-head box protein 3 (FOXP3) regulatory T cells (nTregs) are key mediators of immunity, which orchestrate and maintain tolerance to self and foreign antigens. In the recent 1.5 decades, a multitude of studies have aimed to define the phenotype and function of nTregs and to assess their therapeutic potential for modulating immune mediated disorders such as auto-immunity, allergy, and episodes of transplant rejection. In this review, we summarize the current knowledge on the biology of nTregs. We address the exact definition of nTregs by specific markers and combinations thereof, which is a prerequisite for the state-of-the-art isolation of defined nTreg populations. Furthermore, we discuss the mechanism by which nTregs mediate immunosuppression and how this knowledge might translate into novel therapeutic modalities. With first clinical studies of nTreg-based therapies being finished, questions concerning the reliable sources of nTregs are becoming more and more eminent. Consequently, approaches allowing conversion of CD4+ T cells into nTregs by coculture with antigen-presenting cells, cytokines, and/or pharmacological agents are discussed. In addition, genetic engineering approaches for the generation of antigen-specific nTregs are described. [ABSTRACT FROM AUTHOR]

We describe for the first time fluorescent virus-like particles decorated with biologically active mono- and multisubunit immune receptors of choice and the basic application of such fluorosomes (FSs) to visualize and target immune receptor-ligand interactions. For that purpose, human embryonic kidney (HEK)-293 cells were stably transfected with Moloney murine leukemia virus (MoMLV) matrix protein (MA) GFP fusion constructs. To produce FSs, interleukins (ILs), IL-receptors (IL-Rs), and costimulatory molecules were fused to the glycosyl phosphatidyl inositol anchor acceptor sequence of CD16b and coexpressed along with MoMLV group-specific antigen-polymerase (gag-pol) in MA::GFP+ HEK-293 cells. We show that IL-2 decorated but not control-decorated FSs specifically identify normal and malignant IL-2 receptor-positive (IL-2R+) lymphocytes by flow cytometry. In addition to cytokines and costimulatory molecules, FSs were also successfully decorated with the heterotrimeric IL-2Rs, allowing identification of IL-2+ target cells. Specificity of binding was proven by complete inhibition with nonlabeled, soluble ligands. Moreover, IL-2R FSs efficiently neutralized soluble IL-2 and thus induced unresponsiveness of T cells receiving full activation stimuli via T-cell antigen receptor and CD28. FSs are technically simple, multivalent tools for assessing and blocking mono- and multisubunit immune receptor-ligand interactions with natural constituents in a plasma membrane context. [ABSTRACT FROM AUTHOR]

A cationic AuI N‐heterocyclic carbene complex featuring caffeine‐based ligands has shown potent and selective anticancer effects in ovarian cancer cells. This organogold compound is remarkably stable in physiological conditions. Therefore, the observed therapeutic effects are mainly due to non‐covalent interactions of the gold compound with its pharmacological targets. Among the latter, DNA secondary structures—G‐quadruplexes—play a pivotal role in the mechanisms of anticancer action. More information can be found in the Full Paper by S. M. Meier‐Menches, A. Casini, C. Gerner, et al. on page 15528. [ABSTRACT FROM AUTHOR]