There are compelling reasons to opt for primary human natural killer (NK) cells when validating Ca2+ indicators. 1.) NK cells exhibit a high degree of vulnerability to stressors such as indicator loading or light exposure. 2.) The lack of research on NK Ca2+ signaling underscores the necessity for developing reliable assays. 3.) The increased utilization of NK cell therapies necessitates a more profound comprehension of Ca2+ dependent signal transduction. Consequently, an assay was developed to monitor cytosolic Ca2+ signals in individual NK cells simultaneously with their cytotoxic function against cancer cells. We used this assay to assess the suitability of fura-2, fura-PE3, fura-8, fura-10 or fura-red for quantifying Ca2+ signals in NK cells without compromising their cytotoxic function. In contrast to the widely used fura-2, its red-shifted derivative fura-10 did not interfere with NK cytotoxicity over several hours. It exhibited a superior signal-to-noise ratio and good dynamic range, accompanied by minimal bleaching or leakage. Fura-8 and fura-red also preserved NK cell cytotoxicity, but had other disadvantages compared to fura-10. We successfully used fura-10 to report Ca2+ signals in NK cells from blood donors and patients diagnosed with lymphoma and leukemia over several hours at 37 °C during apoptotic or necrotic killing of different cancer cells (K562, THP1, OCI-AML2, and TMD8). Additionally, we show that fura-10 is well suited to report Ca2+ signals in intact murine pancreatic islets, another stress-sensitive cell preparation. Consequently, fura-10 is an optimal choice for measuring Ca²⁺ in primary human NK cells and other primary cell preparations.

A file containing RPPA and RNAseq datasets, pathway analyses and prediction calculations presented in the main figures.

A file containing supplementary figures 1-8 and the supplementary table S1.

Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells recognize and eliminate cancer cells. However, immune evasion, downregulation of immune function by the tumour microenvironment and resistance of cancer cells are major problems. Although CTL and NK cells are both important to eliminate cancer, most studies address them individually. We quantified sequential primary human CTL and NK cell cytotoxicity against the melanoma cell line SK-Mel-5. At high effector-to-target ratios, NK cells or melan-A (MART-1)-specific CTL eliminated all SK-Mel-5 cells within 24 h, indicating that SK-Mel-5 cells are not resistant initially. However, at lower effector-to-target ratios, which resemble numbers of the immune contexture in human cancer, a substantial number of SK-Mel-5 cells survived. Pre-exposure to CTL induced resistance in surviving SK-Mel-5 cells to subsequent CTL or NK cell cytotoxicity, and pre-exposure to NK cells induced resistance in surviving SK-Mel-5 cells to NK cells. Higher human leucocyte antigen class I expression or interleukin-6 levels were correlated with resistance to NK cells, whereas reduction in MART-1 antigen expression was correlated with reduced CTL cytotoxicity. The CTL cytotoxicity was rescued beyond control levels by exogenous MART-1 antigen. In contrast to the other three combinations, CTL cytotoxicity against SK-Mel-5 cells was enhanced following NK cell pre-exposure. Our assay allows quantification of sequential CTL and NK cell cytotoxicity and might guide strategies for efficient CTL-NK cell anti-melanoma therapies.

The induction of apoptosis is a direct way to eliminate tumor cells and improve cancer therapy. Apoptosis is tightly controlled by the balance of pro- and antiapoptotic Bcl-2 proteins. BH3 mimetics neutralize the antiapoptotic function of Bcl-2 proteins and are highly promising compounds inducing apoptosis in several cancer entities including pediatric malignancies. However, the clinical application of BH3 mimetics in solid tumors is impeded by the frequent resistance to single BH3 mimetics and the anticipated toxicity of high concentrations or combination treatments. One potential avenue to increase the potency of BH3 mimetics is the development of immune cell-based therapies to counteract the intrinsic apoptosis resistance of tumor cells and sensitize them to immune attack. Here, we describe spheroid cultures of pediatric cancer cells that can serve as models for drug testing. In these 3D models, we were able to demonstrate that activated allogeneic Natural Killer (NK) cells migrated into tumor spheroids and displayed cytotoxicity against a wide range of pediatric cancer spheroids, highlighting their potential as anti-tumor effector cells. Next, we investigated whether treatment of tumor spheroids with subtoxic concentrations of BH3 mimetics can increase the cytotoxicity of NK cells. Notably, the cytotoxic effects of NK cells were enhanced by the addition of BH3 mimetics. Treatment with either the Bcl-XL inhibitor A1331852 or the Mcl-1 inhibitor S63845 increased the cytotoxicity of NK cells and reduced spheroid size, while the Bcl-2 inhibitor ABT-199 had no effect on NK cell-mediated killing. Taken together, this is the first study to describe the combination of BH3 mimetics targeting Bcl-XL or Mcl-1 with NK cell-based immunotherapy, highlighting the potential of BH3 mimetics in immunotherapy.

The erythrocyte sedimentation rate (ESR) is one of the oldest medical diagnostic tools. However, currently there is some debate on the structure formed by the cells during the sedimentation process. While the conventional view is that erythrocytes sediment as separate aggregates, others have suggested that they form a percolating gel, similar to other colloidal suspensions. A direct probing of the structures formed by erythrocytes in blood at stasis is then required to settle these discrepancies. Here, we report observations performed with three different optical imaging techniques: direct light transmission through thin samples, two-photon microscopy and light-sheet microscopy. All techniques revealed a dynamic structure of a channeling gel but with differences in the resolved details. A quantitative analysis of the erythrocyte related processes and interactions during the sedimentation need a further refinement of the experimental set-ups.

Abstract Despite impressive advances in melanoma-directed immunotherapies, resistance is common and many patients still succumb to metastatic disease. In this context, harnessing natural killer (NK) cells, which have thus far been sidelined in the development of melanoma immunotherapy, could provide therapeutic benefits for cancer treatment. To identify molecular determinants of NK cell–mediated melanoma killing (NKmK), we quantified NK-cell cytotoxicity against a panel of genetically diverse melanoma cell lines and observed highly heterogeneous susceptibility. Melanoma protein microarrays revealed a correlation between NKmK and the abundance and activity of a subset of proteins, including several metabolic factors. Oxidative phoshorylation, measured by oxygen consumption rate, negatively correlated with melanoma cell sensitivity toward NKmK, and proteins involved in mitochondrial metabolism and epithelial–mesenchymal transition were confirmed to regulate NKmK. Two- and three-dimensional killing assays and melanoma xenografts established that the PI3K/AKT/mTOR signaling axis controls NKmK via regulation of NK cell–relevant surface proteins. A “protein-killing-signature” based on the protein analysis predicted NKmK of additional melanoma cell lines and the response of patients with melanoma to anti-PD-1 checkpoint therapy. Collectively, these findings identify novel NK cell–related prognostic biomarkers and may contribute to improved and personalized melanoma-directed immunotherapies. Significance: NK-cell cytotoxicity assays and protein microarrays reveal novel biomarkers of NK cell–mediated melanoma killing and enable development of signatures to predict melanoma patient responsiveness to immunotherapies.

Visualization cell interaction with the extracellular matrix (ECM) mesh works plays a central role in understanding cell behavior and the corresponding regulatory mechanisms by the environment in vivo. However, long term visualization of 3D matrix structures remains challenging mainly due to photobleaching or blind spot in the currently available approaches. In this paper, we developed a label-free method based on light-sheet microcopy, termed light-sheet scattering microscopy (LSSM), as a satisfactory solution to solve this problem. LSSM can reliably determine structure of collagen matrices from different origin including bovine, human and rat tail. We verified that the quality and intensity of collagen structure images acquired by LSSM did not decline with time. LSSM offers abundant wavelength choice for matrix structure, maximizing combination possibilities for fluorescence to label the cells. LSSM can be used for visualizing ECM-cell interaction in 3D for long term and characterization of cell-applied forces. Interestingly, we observed ultrathin thread-like structures between cells and matrix using LSSM, which was not to be seen by normal fluorescence microscopy. In summary, LSSM provides a robust approach to investigate the complex interplay between cells and ECM in vitro under in vivo-mimicking conditions.

CTL and NK cells recognize and eliminate cancer cells. However, immune evasion, down regulation of immune function by the tumor microenvironment, or resistance of cancer cells are a major problem. While CTL and NK cells are both important to eliminate cancer, most studies address them individually. In a new experimental human model, we analysed combined primary human CTL and NK cell cytotoxicity against the melanoma cell line SK-Mel-5. At high effector-to-target ratios, MART-1-specific CTL or NK cells eliminated SK-Mel-5 cells within 24 hours indicating that SK-Mel-5 cells are initially not resistant. However, at lower effector-to-target ratios, which resemble conditions of the immune contexture in human cancer, a significant number of SK-Mel-5 cells survived. Whereas CTL pre-exposure induced resistance in surviving SK-Mel-5 cells to subsequent CTL or NK cell cytotoxicity, NK cell pre-exposure induced resistance in surviving SK-Mel-5 cells to NK cells but not to MART-1 specific CTL. In contrast, there was even a slight enhancement of CTL cytotoxicity against SK-Mel-5 cells following NK cell pre-exposure. In all other combinations, resistance to subsequent cytotoxicity was higher, if melanoma cells were pre-exposed to larger numbers of CTL or NK cells. Increases in human leukocyte antigen class I expression correlated with resistance to NK cells, while reduction in MART-1 antigen expression correlated with reduced CTL cytotoxicity. CTL cytotoxicity was rescued beyond control levels by exogenous MART-1 antigen. This study quantifies the interdependence of CTL and NK cell cytotoxicity and may guide strategies for efficient CTL-NK cell anti-melanoma therapies. Key points summary Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells eliminate cancer cells. CTL and NK work in parallel, but most studies address them individually. In a new human experimental model, antigen-specific CTL and NK cell cytotoxicity interdependence against melanoma is shown. Whereas high numbers of antigen-specific CTL and NK cells eliminate all melanoma cells, lower, more physiological numbers induce resistance, in case secondary CTL or NK cell exposure follow initial CTL cell exposure or if secondary NK cell exposure follows initial NK cell exposure; only if secondary CTL exposure follows initial NK cell exposure no resistance of melanoma but even a slight enhancement of cytotoxicity was observed. Alterations in HLA-I expression correlated with resistance to NK cells, while reduction in antigen expression correlated with reduced CTL cytotoxicity. CTL cytotoxicity was rescued beyond control levels by exogenous antigen. The results should help to better understand and optimize immune therapies against cancer. Graphical abstract

Immune therapy of cancer is among the most promising recent advances in medicine. Whether the immune system can keep cancer in check depends on, among other factors, the efficiency of immune cells to recognize and eliminate cancer cells. We describe a time-resolved single-cell assay that reports the quality, quantity, and kinetics of target cell death induced by single primary human natural killer (NK) cells. The assay reveals that single NK cells induce cancer cell death by apoptosis and necrosis but also by mixed forms. Inhibition of either one of the two major cytotoxic pathways, perforin/granzyme release or FasL/FasR interaction, unmasked the parallel activity of the other one. Ca2+ influx through Orai channels is important for tuning killer cell function. We found that the apoptosis/necrosis ratio of cancer cell death by NK cells is controlled by the magnitude of Ca2+ entry and furthermore by the relative concentrations of perforin and granzyme B. The possibility to change the apoptosis/necrosis ratio employed by NK cells offers an intriguing possibility to modulate the immunogenicity of the tumor microenvironment.

Key points Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells are required to eliminate cancer cells. We analysed the Ca2+ dependence of CTL and NK cell cytotoxicity and found that in particular CTLs have a very low optimum of [Ca2+]i (between 122 and 334 nm) and [Ca2+]o (between 23 and 625 μm) for efficient cancer cell elimination, well below blood plasma Ca2+ levels. As predicted from these results, partial down‐regulation of the Ca2+ channel Orai1 in CTLs paradoxically increases perforin‐dependent cancer cell killing. Lytic granule release at the immune synapse between CTLs and cancer cells has a Ca2+ optimum compatible with this low Ca2+ optimum for efficient cancer cell killing, whereas the Ca2+ optimum for CTL migration is slightly higher and proliferation increases monotonously with increasing [Ca2+]o. We propose that a partial inhibition of Ca2+ signals by specific Orai1 blockers at submaximal concentrations could contribute to tumour elimination. AbstractCytotoxic T lymphocytes (CTLs) and natural killer (NK) cells are required to protect the human body against cancer. Ca2+ is a key metabolic factor for lymphocyte function and cancer homeostasis. We analysed the Ca2+ dependence of CTL and NK cell cytotoxicity against cancer cells and found that CTLs have a bell‐shaped Ca2+ dependence with an optimum for cancer cell elimination at rather low [Ca2+]o (23–625 μm) and [Ca2+]i (122–334 nm). This finding predicts that a partial inhibition of Orai1 should increase (rather than decrease) cytotoxicity of CTLs at [Ca2+]o higher than 625 μm. We tested this hypothesis in CTLs and indeed found that partial down‐regulation of Orai1 by siRNA increases the efficiency of cancer cell killing. We found two mechanisms that may account for the Ca2+ optimum of cancer cell killing: (1) migration velocity and persistence have a moderate optimum between 500 and 1000 μm [Ca2+]o in CTLs, and (2) lytic granule release at the immune synapse between CTLs and cancer cells is increased at 146 μm compared to 3 or 800 μm, compatible with the Ca2+ optimum for cancer cell killing. It has been demonstrated in many cancer cell types that Orai1‐dependent Ca2+ signals enhance proliferation. We propose that a decrease of [Ca2+]o or partial inhibition of Orai1 activity by selective blockers in the tumour microenvironment could efficiently reduce cancer growth by simultaneously increasing CTL and NK cell cytotoxicity and decreasing cancer cell proliferation. image

Cytotoxic T lymphocytes are effector CD8 + T cells that eradicate infected and malignant cells. Here we show that the transcription factor NFATc1 controls the cytotoxicity of mouse cytotoxic T lymphocytes. Activation of Nfatc1 −/− cytotoxic T lymphocytes showed a defective cytoskeleton organization and recruitment of cytosolic organelles to immunological synapses. These cells have reduced cytotoxicity against tumor cells, and mice with NFATc1-deficient T cells are defective in controlling Listeria infection. Transcriptome analysis shows diminished RNA levels of numerous genes in Nfatc1 −/− CD8 + T cells, including Tbx21 , Gzmb and genes encoding cytokines and chemokines, and genes controlling glycolysis. Nfatc1 −/− , but not Nfatc2 −/− CD8 + T cells have an impaired metabolic switch to glycolysis, which can be restored by IL-2. Genome-wide ChIP-seq shows that NFATc1 binds many genes that control cytotoxic T lymphocyte activity. Together these data indicate that NFATc1 is an important regulator of cytotoxic T lymphocyte effector functions.

Cytotoxic T-lymphocytes (CTLs) and natural killer (NK) cells are the killer cells of the immune system and are responsible for the elimination of potential dangers, such as virus-infected and tumor cells. CTLs and NK cells migrate actively within tissues to encounter target cells, and form a tight connection with them called the immunological synapse that enables killing. However, how migration is regulated in these cells for an efficient reach of their target remains a central question in immunology. Here we analyzed the migration of CTLs and NK cells in 2 dimensions in the absence of external cues to mirror the initial immune response where no signals guide killer cells to their target. By calculating the mean square displacement and the velocity autocorrelation we found that CTLs perform a persistent random walk. Furthermore, cell shape analysis enabled us to establish a model for killer cell migration where the movement of the cell is determined by several independent internal "force generators". Each of them switches between active and inactive states. We found a correlation between the number of the "force generators" and the duration of their activity to the number and life time of lamellipodia. Our numerical simulations revealed that the search time in a given space depends on the behavior of cells upon contact with boundaries. We believe that simulation of migration and search strategy of killer cells will ultimately lead to an improved understanding of the immune response.

Referees 1 and 2 are more positive, still questioning the novelty aspect of the study, the maybe inappropriate use of the MCU inhibitor and the limited provision of mechanistic understanding. Referee 3 (who knowns our journal well) is much more critical and raise serious conceptual issues. For example, the cell line used is not appropriate for looking into metastasis and there seem to be some misconception between migration/invasion and metastasis, especially when the drug used already reduced tumour size. This referee also highlights the study limitations in term of novelty, mechanism and most importantly for our scope, confirms our suspicions that the study lacks medical/clinical validation and pathophysiological insights which we believe, would need more than 3-months to address.

This double-blind, randomized, placebo-controlled, cross-over intervention-study was conducted in healthy volunteers to evaluate the effects of plant sterol ester supplemented margarine on cholesterol, non-cholesterol sterols and oxidative stress in serum and monocytes. Sixteen volunteers, average age 34 years, with no or mild hypercholesterolemia were subjected to a 4 week period of daily intake of 3g plant sterols per day supplied via a supplemented margarine on top of regular eating habits. After a wash-out period of one week, volunteers switched groups. Compared to placebo, a diet supplementation with plant sterols increased serum levels of plant sterols such as campesterol (+0.16±0.19mg/dL, p=0.005) and sitosterol (+0.27±0.18mg/dL, p<0.001) and increased markers of cholesterol synthesis such as desmosterol (+0.05±0.07mg/dL, p=0.006) as well as lathosterol (+0.11±0.16mg/dL, p=0.012). Cholesterol serum levels, however, were not changed significantly (+18.68±32.6mg/dL, p=0.052). These findings could not be verified in isolated circulating monocytes. Moreover, there was no effect on monocyte activation and no differences with regard to redox state after plant sterol supplemented diet. Therefore, in a population of healthy volunteers with no or mild hypercholesterolemia, consumption of plant sterol ester supplemented margarine results in increased concentrations of plant sterols and cholesterol synthesis markers without affecting total cholesterol in the serum, activation of circulating monocytes or redox state.

Triple-negative breast cancer (TNBC) represents the most aggressive breast tumor subtype. However, the molecular determinants responsible for the metastatic TNBC phenotype are only partially understood. We here show that expression of the mitochondrial calcium uniporter (MCU), the selective channel responsible for mitochondrial Ca(2+) uptake, correlates with tumor size and lymph node infiltration, suggesting that mitochondrial Ca(2+) uptake might be instrumental for tumor growth and metastatic formation. Accordingly, MCU downregulation hampered cell motility and invasiveness and reduced tumor growth, lymph node infiltration, and lung metastasis in TNBC xenografts. In MCU-silenced cells, production of mitochondrial reactive oxygen species (mROS) is blunted and expression of the hypoxia-inducible factor-1α (HIF-1α) is reduced, suggesting a signaling role for mROS and HIF-1α, downstream of mitochondrial Ca(2+) Finally, in breast cancer mRNA samples, a positive correlation of MCU expression with HIF-1α signaling route is present. Our results indicate that MCU plays a central role in TNBC growth and metastasis formation and suggest that mitochondrial Ca(2+) uptake is a potential novel therapeutic target for clinical intervention.

Background: The function of formyl peptide receptors (FPRs) is incompletely understood. Results: We report the identification of bacterial signal peptides as potent activators of mammalian FPRs and innate immune responses and define critical features underlying FPR peptide recognition. Conclusion: These findings identify a molecular signature for FPR activation. Significance: Our results define a novel mechanism for sensing bacteria. Formyl peptide receptors (FPRs) are G-protein-coupled receptors that function as chemoattractant receptors in innate immune responses. Here we perform systematic structure-function analyses of FPRs from six mammalian species using structurally diverse FPR peptide agonists and identify a common set of conserved agonist properties with typical features of pathogen-associated molecular patterns. Guided by these results, we discover that bacterial signal peptides, normally used to translocate proteins across cytoplasmic membranes, are a vast family of natural FPR agonists. N-terminally formylated signal peptide fragments with variable sequence and length activate human and mouse FPR1 and FPR2 at low nanomolar concentrations, thus establishing FPR1 and FPR2 as sensitive and broad signal peptide receptors. The vomeronasal receptor mFpr-rs1 and its sequence orthologue hFPR3 also react to signal peptides but are much more narrowly tuned in signal peptide recognition. Furthermore, all signal peptides examined here function as potent activators of the innate immune system. They elicit robust, FPR-dependent calcium mobilization in human and mouse leukocytes and trigger a range of classical innate defense mechanisms, such as the production of reactive oxygen species, metalloprotease release, and chemotaxis. Thus, bacterial signal peptides constitute a novel class of immune activators that are likely to contribute to mammalian immune defense against bacteria. This evolutionarily conserved detection mechanism combines structural promiscuity with high specificity and enables discrimination between bacterial and eukaryotic signal sequences. With at least 175,542 predicted sequences, bacterial signal peptides represent the largest and structurally most heterogeneous class of G-protein-coupled receptor agonists currently known for the innate immune system.

Department of Physiology, Saarland University Faculty of Medicine, Homburg, Germany Key words: CTL; cytotoxicity; killing kinetics; real-time assay; NK cells. Corresponding author: Dr. Bin Qu Institute of Biophysics Saarland University Building 58 66421 Homburg Germany Tel.: +49-6841-162-6458 Fax: +49-6841-162-6060 bin.qu@uks.eu