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Abstract Upon stimulation of membrane receptors, nicotinic acid adenine dinucleotide phosphate (NAADP) is formed as second messenger within seconds and evokes Ca2+ signaling in many different cell types. Here, to directly stimulate NAADP signaling, MASTER-NAADP, a Membrane permeAble, STabilized, bio-rEversibly pRotected precursor of NAADP is synthesized and release of its active NAADP mimetic, benzoic acid C-nucleoside, 2’-phospho-3’F-adenosine-diphosphate, by esterase digestion is confirmed. In the presence of NAADP receptor HN1L/JPT2 (hematological and neurological expressed 1-like protein, HN1L, also known as Jupiter microtubule-associated homolog 2, JPT2), this active NAADP mimetic releases Ca2+ and increases the open probability of type 1 ryanodine receptor. When added to intact cells, MASTER-NAADP initially evokes single local Ca2+ signals of low amplitude. Subsequently, also global Ca2+ signaling is observed in T cells, natural killer cells, and Neuro2A cells. In contrast, control compound MASTER-NADP does not stimulate Ca2+ signaling. Likewise, in cells devoid of HN1L/JPT2, MASTER-NAADP does not affect Ca2+ signaling, confirming that the product released from MASTER-NAADP is a bona fide NAADP mimetic.

CD39 is the major enzyme controlling the levels of extracellular adenosine triphosphate (ATP) via the stepwise hydrolysis of ATP to adenosine diphosphate (ADP) and adenosine monophosphate (AMP). As extracellular ATP is a strong promoter of inflammation, monoclonal antibodies (mAbs) blocking CD39 are utilized therapeutically in the field of immune-oncology. Though anti-CD39 mAbs are highly specific for their target, they lack deep penetration into the dense tissue of solid tumors, due to their large size. To overcome this limitation, we generated and characterized nanobodies that targeted and blocked human CD39. From cDNA-immunized alpacas we selected 16 clones from seven nanobody families that bind to two distinct epitopes of human CD39. Among these, clone SB24 inhibited the enzymatic activity of CD39. Of note, SB24 blocked ATP degradation by both soluble and cell surface CD39 as a 15kD monomeric nanobody. Dimerization via fusion to an immunoglobulin Fc portion further increased the blocking potency of SB24 on CD39-transfected HEK cells. Finally, we confirmed the CD39 blocking properties of SB24 on human PBMCs. In summary, SB24 provides a new small biological antagonist of human CD39 with potential application in cancer therapy.

CD8+ T cells are a crucial part of the adaptive immune system, responsible for combating intracellular pathogens and tumor cells. The initial activation of T cells involves the formation of highly dynamic Ca2+ microdomains. Recently, purinergic signaling was shown to be involved in the formation of the initial Ca2+ microdomains in CD4+ T cells. In this study, the role of purinergic cation channels, particularly P2X4 and P2X7, in CD8+ T cell signaling from initial events to downstream responses was investigated, focusing on various aspects of T cell activation, including Ca2+ microdomains, global Ca2+ responses, NFAT-1 translocation, cytokine expression, and proliferation. While Ca2+ microdomain formation was significantly reduced in the first milliseconds to seconds in CD8+ T cells lacking P2X4 and P2X7 channels, global Ca2+ responses over minutes were comparable between wild-type (WT) and knockout cells. However, the onset velocity was reduced in P2X4-deficient cells, and P2X4, as well as P2X7-deficient cells, exhibited a delayed response to reach a certain level of free cytosolic Ca2+ concentration ([Ca2+]i). NFAT-1 translocation, a crucial transcription factor in T cell activation, was also impaired in CD8+ T cells lacking P2X4 and P2X7. In addition, the expression of IFN-γ, a major pro-inflammatory cytokine produced by activated CD8+ T cells, and Nur77, a negative regulator of T cell activation, was significantly reduced 18h post-stimulation in the knockout cells. In line, the proliferation of T cells after 3 days was also impaired in the absence of P2X4 and P2X7 channels. In summary, the study demonstrates that purinergic signaling through P2X4 and P2X7 enhances initial Ca2+ events during CD8+ T cell activation and plays a crucial role in regulating downstream responses, including NFAT-1 translocation, cytokine expression, and proliferation on multiple timescales. These findings suggest that targeting purinergic signaling pathways may offer potential therapeutic interventions.

IntroductionDuring thermogenesis, adipose tissue (AT) becomes more active and enhances oxidative metabolism. The promotion of this process in white AT (WAT) is called “browning” and, together with the brown AT (BAT) activation, is considered as a promising approach to counteract obesity and metabolic diseases. Transient receptor potential cation channel, subfamily M, member 2 (TRPM2), is an ion channel that allows extracellular Ca2+ influx into the cytosol, and is gated by adenosine diphosphate ribose (ADPR), produced from NAD+ degradation. The aim of this study was to investigate the relevance of TRPM2 in the regulation of energy metabolism in BAT, WAT, and liver during thermogenesis.MethodsWild type (WT) and Trpm2-/- mice were exposed to 6°C and BAT, WAT and liver were collected to evaluate mRNA, protein levels and ADPR content. Furthermore, O2 consumption, CO2 production and energy expenditure were measured in these mice upon thermogenic stimulation. Finally, the effect of the pharmacological inhibition of TRPM2 was assessed in primary adipocytes, evaluating the response upon stimulation with the β-adrenergic receptor agonist CL316,243.ResultsTrpm2-/- mice displayed lower expression of browning markers in AT and lower energy expenditure in response to thermogenic stimulus, compared to WT animals. Trpm2 gene overexpression was observed in WAT, BAT and liver upon cold exposure. In addition, ADPR levels and mono/poly-ADPR hydrolases expression were higher in mice exposed to cold, compared to control mice, likely mediating ADPR generation.DiscussionOur data indicate TRPM2 as a fundamental player in BAT activation and WAT browning. TRPM2 agonists may represent new pharmacological strategies to fight obesity.

Ca2+ microdomains play a key role in intracellular signaling processes. For instance, they mediate the activation of T cells and, thus, the initial adaptive immune system. They are, however, also of utmost importance for activation of other cells, and a detailed understanding of the dynamics of these spatially localized Ca2+ signals is crucial for a better understanding of the underlying signaling processes. A typical approach to analyze Ca2+ microdomain dynamics is live cell fluorescence microscopy imaging. Experiments usually involve imaging a larger number of cells of different groups (for instance, wild type and knockout cells), followed by a time consuming image and data analysis. With DARTS, we present a modular Python pipeline for efficient Ca2+ microdomain analysis in live cell imaging data. DARTS (Deconvolution, Analysis, Registration, Tracking, and Shape normalization) provides state-of-the-art image postprocessing options like deep learning-based cell detection and tracking, spatio-temporal image deconvolution, and bleaching correction. An integrated automated Ca2+ microdomain detection offers direct access to global statistics like the number of microdomains for cell groups, corresponding signal intensity levels, and the temporal evolution of the measures. With a focus on bead stimulation experiments, DARTS provides a so-called dartboard projection analysis and visualization approach. A dartboard projection covers spatio-temporal normalization of the bead contact areas and cell shape normalization onto a circular template that enables aggregation of the spatiotemporal information of the microdomain detection results for the individual cells of the cell groups of interest. The dartboard visualization allows intuitive interpretation of the spatio-temporal microdomain dynamics at the group level. The application of DARTS is illustrated by three use cases in the context of the formation of initial Ca2+ microdomains after cell stimulation. DARTS is provided as an open-source solution and will be continuously extended upon the feedback of the community.Code available at:10.5281/zenodo.10459243.

TRPM2 is a Ca2+ permeable, non-selective cation channel in the plasma membrane that is involved in the innate immune response regulating, for example, chemotaxis in neutrophils and cytokine secretion in monocytes and macrophages. The intracellular adenine nucleotides ADP-ribose (ADPR) and 2′-deoxy-ADPR (2dADPR) activate the channel, in combination with their co-agonist Ca2+. Interestingly, activation of human TRPM2 (hsTRPM2) by 2dADPR is much more effective than activation by ADPR. However, the underlying mechanism of the nucleotides’ differential effect on the channel is not yet fully understood. In this study, we performed whole-cell patch clamp experiments with HEK293 cells heterologously expressing hsTRPM2. We show that 2dADPR has an approx. 4-fold higher Ca2+ sensitivity than ADPR (EC50 = 190 and 690 nM). This allows 2dADPR to activate the channel at lower and thus physiological intracellular Ca2+ concentrations. Kinetic analysis of our data reveals that activation by 2dADPR is faster than activation by ADPR. Mutation in a calmodulin binding N-terminal IQ-like motif in hsTRPM2 completely abrogated channel activation by both agonists. However, mutation of a single amino acid residue (W1355A) in the C-terminus of hsTRPM2, at a site of extensive inter-domain interaction, resulted in slower activation by 2dADPR and neutralized the difference in rate of activation between the two agonists. Taken together, we propose a mechanism by which 2dADPR induces higher hsTRPM2 currents than ADPR by means of faster channel activation. The finding that 2dADPR has a higher Ca2+ sensitivity than ADPR may indicate that 2dADPR rather than ADPR activates hsTRPM2 in physiological contexts such as the innate immune response.

Cellular Ca2+ signaling is highly organized in time and space. Locally restricted and short-lived regions of Ca2+ increase, called Ca2+ microdomains, constitute building blocks that are differentially arranged to create cellular Ca2+ signatures controlling physiological responses. Here, we focus on Ca2+ microdomains occurring in restricted cytosolic spaces between the plasma membrane and the endoplasmic reticulum, called endoplasmic reticulum-plasma membrane junctions. In T cells, these microdomains have been finely characterized. Enough quantitative data are thus available to develop detailed computational models of junctional Ca2+ dynamics. Simulations are able to predict the characteristics of Ca2+ increases at the level of single channels and in junctions of different spatial configurations, in response to various signaling molecules. Thanks to the synergy between experimental observations and computational modeling, a unified description of the molecular mechanisms that create Ca2+ microdomains in the first seconds of T cell stimulation is emerging.

Poly-functional helper T cells can have a stronger effect than mono-functional T cells, but whether the response is qualitatively different is not clear. Here the authors show that a population of IL-17+IL-22+, but not single IL-22+, CD4+ T cells are induced by TGF-β, enriched in patients with colorectal cancer (CRC) and drive CRC progression in mice.

Ca2+ signalling plays an essential role in T cell activation, which is a key step to start an adaptive immune response. During the transition from a quiescent to a fully activated state, Ca2+ microdomains of reduced spatial and temporal extents develop in the junctions between the plasma membrane and the endoplasmic reticulum (ER). These microdomains rely on Ca2+ entry from the extracellular medium, via the ORAI1/STIM1/STIM2 system that mediates store operated Ca2+ entry Store operated calcium entry. The mechanism leading to local store depletion and subsequent Ca2+ entry depends on the activation state of the cells. The initial, smaller microdomains are triggered by D-myo-inositol 1,4,5-trisphosphate (IP3) signalling in response to T cell adhesion. T cell receptor (TCR)/CD3 stimulation then initiates nicotinic acid adenine dinucleotide phosphate signalling, which activates ryanodine receptors (RYR). We have recently developed a mathematical model to elucidate the spatiotemporal Ca2+ dynamics of the microdomains triggered by IP3 signalling in response to T cell adhesion (Gil et al., 2021). This reaction-diffusion model describes the evolution of the cytosolic and endoplasmic reticulum Ca2+ concentrations in a three-dimensional ER-PM junction and was solved using COMSOL Multiphysics. Modelling predicted that adhesion-dependent microdomains result from the concerted activity of IP3 receptors and pre-formed ORAI1-STIM2 complexes. In the present study, we extend this model to include the role of RYRs rapidly after TCR/CD3 stimulation. The involvement of STIM1, which has a lower KD for Ca2+ than STIM2, is also considered. Detailed 3D spatio-temporal simulations show that these Ca2+ microdomains rely on the concerted opening of ∼7 RYRs that are simultaneously active in response to the increase in NAADP induced by T cell stimulation. Opening of these RYRs provoke a local depletion of ER Ca2+ that triggers Ca2+ flux through the ORAI1 channels. Simulations predict that RYRs are most probably located around the junction and that the increase in junctional Ca2+ concentration results from the combination between diffusion of Ca2+ released through the RYRs and Ca2+ entry through ORAI1 in the junction. The computational model moreover provides a tool allowing to investigate how Ca2+ microdomains occur, extend and interact in various states of T cell activation.

Nicotinamide adenine dinucleotide (NAD) and its 2′-phosphorylated cousin NADP are precursors for the enzymatic formation of the Ca2+-mobilizing second messengers adenosine diphosphoribose (ADPR), 2′-deoxy-ADPR, cyclic ADPR, and nicotinic acid adenine dinucleotide phosphate (NAADP). The enzymes involved are either NAD glycohydrolases CD38 or sterile alpha toll/interleukin receptor motif containing-1 (SARM1), or (dual) NADPH oxidases (NOX/DUOX). Enzymatic function(s) are reviewed and physiological role(s) in selected cell systems are discussed.

Antigen recognition by the T-cell receptor induces a cytosolic Ca2+ signal that is crucial for T-cell function. The Ca2+ channel TRPM2 (transient receptor potential cation channel subfamily M member 2) has been shown to facilitate influx of extracellular Ca2+ through the plasma membrane of T cells. Therefore, it was suggested that TRPM2 is involved in T-cell activation and differentiation. However, these results are largely derived from in vitro studies using T-cell lines and non-physiologic means of TRPM2 activation. Thus, the relevance of TRPM2-mediated Ca2+ signaling in T cells remains unclear. Here, we use TRPM2-deficient mice to investigate the function of TRPM2 in T-cell activation and differentiation. In response to TCR stimulation in vitro, Trpm2-/- and WT CD4+ and CD8+ T cells similarly upregulated the early activation markers NUR77, IRF4, and CD69. We also observed regular proliferation of Trpm2-/- CD8+ T cells and unimpaired differentiation of CD4+ T cells into Th1, Th17, and Treg cells under specific polarizing conditions. In vivo, Trpm2-/- and WT CD8+ T cells showed equal specific responses to Listeria monocytogenes after infection of WT and Trpm2-/- mice and after transfer of WT and Trpm2-/- CD8+ T cells into infected recipients. CD4+ T-cell responses were investigated in the model of anti-CD3 mAb-induced intestinal inflammation, which allows analysis of Th1, Th17, Treg, and Tr1-cell differentiation. Here again, we detected similar responses of WT and Trpm2-/- CD4+ T cells. In conclusion, our results argue against a major function of TRPM2 in T-cell activation and differentiation.

Boosting NAD+ levels are considered a promising means to promote healthy aging and ameliorate dysfunctional metabolism. The expression of CD38, the major NAD+-consuming enzyme, is downregulated during thermogenesis in both brown and white adipose tissues (BAT and WAT). Moreover, BAT activation and WAT “browning” were enhanced in Cd38−/− mice. In this study, the role of CD38 in the liver during thermogenesis was investigated, with the liver being the central organ controlling systemic energy metabolism. Wild-type mice and Cd38−/− mice were exposed to cold temperatures, and levels of metabolites and enzymes were measured in the livers and plasma. During cold exposure, CD38 expression was downregulated in the liver, as in BAT and WAT, with a concomitant increase in NAD(H) and a marked decrease in NADPH levels. Glucose-6-phosphate dehydrogenase and the malic enzyme, along with enzymes in the glycolytic pathway, were downregulated, which is in line with glucose-6-P being re-directed towards glucose release. In Cd38−/− mice, the cross-regulation between glycolysis and glucose release was lost, although this did not impair the glucose release from glycogen. Glycerol levels were decreased in the liver from Cd38−/− animals upon cold exposure, suggesting that glyceroneogenesis, as gluconeogenesis, was not properly activated in the absence of CD38. SIRT3 activity, regulating mitochondrial metabolism, was enhanced by cold exposure, whereas its activity was already high at a warm temperature in Cd38−/− mice and was not further increased by the cold. Notably, FGF21 and bile acid release was enhanced in the liver of Cd38−/− mice, which might contribute to enhanced BAT activation in Cd38−/− mice. These results demonstrate that CD38 inhibition can be suggested as a strategy to boost NAD+ and would not negatively affect hepatic functions during thermogenesis.

Nicotinic acid adenine dinucleotide 2’-phosphate (NAADP) is a naturally occurring nucleotide that has been shown to be involved in the release of Ca2+ from intracellular stores in a wide variety of cell types, tissues and organisms. Current evidence suggests that NAADP may function as a trigger to initiate a Ca2+ signal that is then amplified by other Ca2+ release mechanisms. A fundamental question that remains unanswered is the identity of the NAADP receptor. Our recent studies have identified HN1L/JPT2 as a high affinity NAADP binding protein that is essential for the modulation of Ca2+ channels.

Ca2+ signaling plays an essential role in T cell activation, which is a key step to start an adaptive immune response. During the transition from a quiescent to a fully activated state, Ca2+ microdomains characterized by reduced spatial and temporal extents are observed in the junctions between the plasma membrane (PM) and the endoplasmic reticulum (ER). Such Ca2+ responses can also occur in response to T cell adhesion to other cells or extracellular matrix proteins in otherwise unstimulated T cells. These non-TCR/CD3-dependent Ca2+ microdomains rely on d-myo-inositol 1,4,5-trisphosphate (IP3) signaling and subsequent store operated Ca2+ entry (SOCE) via the ORAI/STIM system. The detailed molecular mechanism of adhesion-dependent Ca2+ microdomain formation remains to be fully elucidated. We used mathematical modeling to investigate the spatiotemporal characteristics of T cell Ca2+ microdomains and their molecular regulators. We developed a reaction-diffusion model using COMSOL Multiphysics to describe the evolution of cytosolic and ER Ca2+ concentrations in a three-dimensional ER-PM junction. Equations are based on a previously proposed realistic description of the junction, which is extended to take into account IP3 receptors (IP3R) that are located next to the junction. The first model only considered the ORAI channels and the SERCA pumps. Taking into account the existence of preformed clusters of ORAI1 and STIM2, ORAI1 slightly opens in conditions of a full ER. These simulated Ca2+ microdomains are too small as compared to those observed in unstimulated T cells. When considering the opening of the IP3Rs located near the junction, the local depletion of ER Ca2+ allows for larger Ca2+ fluxes through the ORAI1 channels and hence larger local Ca2+ concentrations. Computational results moreover show that Ca2+ diffusion in the ER has a major impact on the Ca2+ changes in the junction, by affecting the local Ca2+ gradients in the sub-PM ER. Besides pointing out the likely involvement of the spontaneous openings of IP3Rs in the activation of SOCE in conditions of T cell adhesion prior to full activation, the model provides a tool to investigate how Ca2+ microdomains extent and interact in response to T cell receptor activation.

Natural killer (NK) cells are an important component of the innate immune system for the control of intracellular pathogens and cancer cells. NK cells demonstrate heterogeneous expression of inhibitory surface receptors. Signaling through these various receptors during NK cell development promotes functionality, referred to as NK cell education. Here we investigated the impact of education on NK cell metabolism through functional assessment of critical metabolic pathways and calcium signaling. Educated NK cells had an increased uptake of the metabolic substrates 2-NBDG, a fluorescent glucose analog, and BODIPY FL C16, a fluorescent palmitate, compared to uneducated NK cells. Comparison of NK cells educated via KIRs or NKG2A showed that NKG2A-educated NK cells were the main contributor to these differences in uptake of metabolites, and that NKG2A-educated NK cells were functionally more resilient in response to metabolic blockade of oxidative phosphorylation. Furthermore, NKG2A-educated NK cells exhibited higher peak calcium concentration following stimulation, indicating stronger signaling events taking place in these educated NK cells. These results demonstrate that cellular metabolism plays an important role in the functional differences observed between educated and uneducated NK cells, and show that NKG2A-educated NK cells remain more functionally competent than KIR-educated NK cells when oxidative phosphorylation is restricted. Understanding metabolic programming during NK cell education may unveil future targets to manipulate NK cell function for use in clinical settings, such as cancer therapies.

TRPM2 is a non-selective, Ca2+-permeable cation channel widely expressed in immune cells. It is firmly established that the channel can be activated by intracellular adenosine 5′-diphosphoribose (ADPR). Until recent cryo-EM structures have exhibited an additional nucleotide binding site in the N-terminus of the channel, this activation was thought to occur via binding to a C-terminal domain of the channel that is highly homologous to the ADPR pyrophosphatase NudT9. Over the years it has been controversially discussed whether the Ca2+ mobilizing second messenger cyclic ADP ribose (cADPR) might also directly activate Ca2+ entry via TRPM2. Here we will review the status of this discussion.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a second messenger that evokes calcium release from intracellular organelles by the engagement of calcium release channels, including members of the Transient Receptor Potential (TRP) family, such as TRPML1, the (structurally) related Two Pore Channel type 1 (TPC1) and TPC2 channels as well as Ryanodine Receptors type 1 (RYR1; Guse, 2012). NAADP evokes calcium release from acidic calcium stores of many cell types (Guse, 2012), and NAADP-sensitive Ca2+ stores have been described in hippocampal neurons of the rat (Bak et al., 1999; McGuinness et al., 2007). Glutamate triggers Ca2+-mediated neuronal excitotoxicity in inflammation-induced neurodegenerative pathologies such as Multiple Sclerosis (MS; Friese et al., 2014), and when applied extracellularly to neurons glutamate can elevate NAADP levels in these cells. Accordingly, glutamate-evoked Ca2+ signals from intracellular organelles were inhibited by preventing organelle acidification (Pandey et al., 2009). Analysis of reported RNA sequencing experiments of cultured hippocampal neurons revealed the abundance of Mcoln1 (encoding TRPML1), Tpcn1, and Tpcn2 (encoding TPC1 and TPC2, respectively) as potential NAADP target channels in these cells. Transcripts encoding Ryr1 were not found in contrast to Ryr2 and Ryr3. To study the contribution of NAADP signaling to glutamate-evoked calcium transients in murine hippocampal neurons we used the NAADP antagonists Ned-19 (Naylor et al., 2009) and BZ194 (Dammermann et al., 2009). Our results show that both NAADP antagonists significantly reduce glutamate-evoked calcium transients. In addition to extracellular glutamate application, we studied synchronized calcium oscillations in the cells of the neuronal cultures evoked by addition of the GABAA receptor antagonist bicuculline. Pretreatment with Ned-19 (50 μM) or BZ194 (100 μM) led to an increase in the frequency of bicuculline-induced calcium oscillations at the cost of calcium transient amplitudes. Interestingly, Ned-19 triggered a rise in intracellular calcium concentrations 25 min after bicuculline stimulation, leading to the question whether NAADP acts as a neuroprotective messenger in hippocampal neurons. Taken together, our results are in agreement with the concept that NAADP signaling significantly contributes to glutamate evoked Ca2+ rise in hippocampal neurons and to the amplitude and frequency of synchronized Ca2+ oscillations triggered by spontaneous glutamate release events.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a universal Ca2+ mobilizing second messenger essential for initiation of Ca2+ signaling. Recently, novel molecular mechanisms of both its rapid formation upon receptor stimulation and its mode of action were discovered. Dual NADPH oxidase 2 (DUOX2) and hematological and neurological expressed 1-like protein (HN1L)/Jupiter microtubule-associated homolog 2 (JPT2) were discovered as NAADP-forming enzyme and NAADP receptor/binding protein—the new kids on the block. These novel aspects are reviewed and integrated into the previous view of NAADP signaling.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent Ca2+ mobilizing agent and its inhibition proved to inhibit T-cell activation. However, the impact of the NAADP signaling on CD4+ T-cell differentiation and plasticity and on the inflammation in tissues other than the central nervous system remains unclear. In this study, we used an antagonist of NAADP signaling, trans-Ned 19, to study the role of NAADP in CD4+ T-cell differentiation and effector function. Partial blockade of NAADP signaling in naïve CD4+ T cells in vitro promoted the differentiation of Th17 cells. Interestingly, trans-Ned 19 also promoted the production of IL-10, co-expression of LAG-3 and CD49b and increased the suppressive capacity of Th17 cells. Moreover, using an IL-17A fate mapping mouse model, we showed that NAADP inhibition promotes conversion of Th17 cells into regulatory T cells in vitro and in vivo. In line with the results, we found that inhibiting NAADP ameliorates disease in a mouse model of intestinal inflammation. Thus, these results reveal a novel function of NAADP in controlling the differentiation and plasticity of CD4+ T cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Ca2+-mobilizing adenine nucleotide second messengers cyclic adenosine diphosphoribose, (cADPR), nicotinic acid adenine dinucleotide phosphate (NAADP), adenosine diphosphoribose (ADPR), and 2′deoxy-ADPR were discovered since the late 1980s. They either release Ca2+ from endogenous Ca2+ stores, e.g., endoplasmic reticulum or acidic organelles, or evoke Ca2+ entry by directly activating a Ca2+ channel in the plasma membrane. For 25 years, Professor Barry Potter has been one of the major medicinal chemists in this topical area, designing and contributing numerous analogues to develop structure–activity relationships (SAR) as a basis for tool development in biochemistry and cell biology and for lead development in proof-of-concept studies in disease models. With this review, I wish to acknowledge our 25-year-long collaboration on Ca2+-mobilizing adenine nucleotide second messengers as a major part of Professor Potter’s scientific lifetime achievements on the occasion of his retirement in 2020.

Early Ca2+ signaling is characterized by occurrence of Ca2+ microdomains formed by opening of single or clusters of Ca2+ channels, thereby initiating first signaling and subsequently activating global Ca2+ signaling mechanisms. However, only few data are available focusing on the first seconds and minutes of Ca2+ microdomain formation and related signaling pathways in activated T-lymphocytes. In this review, we condense current knowledge on Ca2+ microdomain formation in T-lymphocytes and early Ca2+ signaling, function of Ca2+ microdomains, and microdomain organization. Interestingly, considering the first seconds of T cell activation, a triphasic Ca2+ signal is becoming apparent: (i) initial Ca2+ microdomains occurring in the first second of T cell activation, (ii) amplification of Ca2+ microdomains by recruitment of further channels in the next 5–10 s, and (iii) a transition to global Ca2+ increase. Apparently, the second messenger nicotinic acid adenine dinucleotide phosphate is the first second messenger involved in initiation of Ca2+ microdomains. Ryanodine receptors type 1 act as initial Ca2+ release channels in CD4+ T-lymphocytes. Regarding the temporal correlation of Ca2+ microdomains with other molecular events of T cell activation, T cell receptor-dependent microdomain organization of signaling molecules Grb2 and Src homology [SH2] domain-containing leukocyte protein of 65 kDa was observed within the first 20 s. In addition, fast cytoskeletal changes are initiated. Furthermore, the involvement of additional Ca2+ channels and organelles, such as the Ca2+ buffering mitochondria, is discussed. Future research developments will comprise analysis of the causal relation between these temporally coordinated signaling events. Taken together, high-resolution Ca2+ imaging techniques applied to T cell activation in the past years paved the way to detailed molecular understanding of initial Ca2+ signaling mechanisms in non-excitable cells.

Adenine nucleotide (AN) 2nd messengers, such as 3′,5′-cyclic adenosine monophosphate (cAMP), are central elements of intracellular signaling, but many details of their underlying processes remain elusive. Like all nucleotides, cyclic nucleotide monophosphates (cNMPs) are net-negatively charged at physiologic pH which limits their applicability in cell-based settings. Thus, many cellular assays rely on sophisticated techniques like microinjection or electroporation. This setup is not feasible for medium- to high-throughput formats, and the mechanic stress that cells are exposed to raises the probability of interfering artefacts or false-positives. Here, we present a short and flexible chemical route yielding membrane-permeable, bio-reversibly masked cNMPs for which we employed the octanoyloxybenzyl (OB) group. We further show hydrolysis studies on chemical stability and enzymatic activation, and present results of real-time assays, where we used cAMP and Ca2+ live cell imaging to demonstrate high permeability and prompt intracellular conversion of some selected masked cNMPs. Based on these results, our novel OB-masked cNMPs constitute valuable precursor-tools for non-invasive studies on intracellular signaling.

OBJECTIVES:Choosing the right mentor is crucial for effective mentorship. Yet, many medical students have difficulties finding a suitable mentor. Thus we developed mentoring speed dating (MSD) as a promising matching tool to connect students and faculty mentors successfully. The purpose of this study was to explore mentees' and mentors' experience with MSD and investigate the impact of MSD on the perceived mentorship quality and continuance of the mentoring relationship. METHODS:The authors completed a mixed methods study at the University Medical Center Hamburg-Eppendorf, Germany, between June 2011 and March 2014. They conducted four focus groups with mentees and mentors who participated in a mentoring speed dating event and analyzed transcripts using conventional content analysis with inductive categorizing. In addition, three mentoring cohorts (two matched via MSD, one matched via conventional online profiles) were surveyed on mentorship satisfaction and the 1-year continuance of their mentorship was monitored. Fifteen mentees and fifteen mentors participated in the focus groups. The authors identified several themes such as short and long term benefits of MSD and fulfillment of expectations. Benefits included finding out about the personal connection, matching expectations, providing an efficient overview of candidates. The survey was completed by 93 students (n = 29 without MSD; n = 64 with MSD). Independent t-tests and multivariate analysis of variance were used to analyze the impact of MSD on student's mentorship satisfaction. RESULTS:There were significant differences in responses to the items "Commitment of mentor" (p = .019) and "Constructive feedback" (p = .038) among the students who attended MSD and the students without MSD. After one year far more mentoring relationships existed among those mentees who participated in MSD in comparison to the "no MSD group". CONCLUSION:MSD is a valuable matching tool with beneficial effects on the mentorship quality. It enhances essential factors in the mentoring relationship such as commitment and satisfaction.

Fate decision processes of T lymphocytes are crucial for health and disease. Whether a T lymphocyte is activated, divides, gets anergic or initiates apoptosis depends on extracellular triggers and intracellular signalling. Free cytosolic calcium dynamics plays an important role in this context. The relative contributions of store-derived calcium entry and calcium entry from extracellular space to T lymphocyte activation are still a matter of debate. Here we develop a quantitative mathematical model of T lymphocyte calcium dynamics in order to establish a tool which allows to disentangle cause-effect relationships between ion fluxes and observed calcium time courses. The model is based on single transmembrane protein characteristics which have been determined in independent experiments. This reduces the number of unknown parameters in the model to a minimum and ensures the predictive power of the model. Simulation results are subsequently used for an analysis of whole cell calcium dynamics measured under various experimental conditions. The model accounts for a variety of these conditions, which supports the suitability of the modelling approach. The simulation results suggest a model in which calcium dynamics dominantly relies on the opening of channels in calcium stores while calcium entry through calcium-release activated channels (CRAC) is more associated with the maintenance of the T lymphocyte calcium levels and prevents the cell from calcium depletion. Our findings indicate that CRAC guarantees a long-term stable calcium level which is required for cell survival and sustained calcium enhancement.

Calcium signaling is a universal signal transduction mechanism in animal and plant cells. In mammalian T-lymphocytes calcium signaling is essential for activation and re-activation and thus important for a functional immune response. Since many years it has been known that both calcium release from intracellular stores and calcium entry via plasma membrane calcium channels are involved in shaping spatio-temporal calcium signals. Second messengers derived from the adenine dinucleotides NAD and NADP have been implicated in T cell calcium signaling. Nicotinic acid adenine dinucleotide phosphate (NAADP) acts as a very early second messenger upon T cell receptor/CD3 engagement, while cyclic ADP-ribose (cADPR) mainly is involved in sustained partial depletion of the endoplasmic reticulum by stimulating calcium release via ryanodine receptors. Finally, adenosine diphosphoribose (ADPR) a breakdown product of both NAD and cADPR activates a plasma membrane cation channel termed TRPM2 thereby facilitating calcium (and sodium) entry into T cells.Receptor-mediated formation, metabolism and mode of action of these novel second messengers in T-lymphocytes will be reviewed.