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Describes a process which may be the mechanism by which electrically nonexcitable cells maintain raised intracellular Ca2+ concentrations and replenish their empty Ca2+ stores after receptor stimulation. Combination of patch-clamp and Fura-2 measurements to monitor membrane currents in mast cells under conditions where intracellular Ca2+ stores were emptied by either inositol 1,4,5-triphosphate, ionomycin, or excess of the Ca2+ chelator EGTA.

Natural killer (NK) cells play a vital role in eliminating tumorigenic cells. Efficient locating and killing of target cells in complex three-dimensional (3D) environments are critical for their functions under physiological conditions. However, the role of mechanosensing in regulating NK-cell killing efficiency in physiologically relevant scenarios is poorly understood. Here, we report that the responsiveness of NK cells is regulated by tumor cell stiffness. NK-cell killing efficiency in 3D is impaired against softened tumor cells, whereas it is enhanced against stiffened tumor cells. Notably, the durations required for NK-cell killing and detachment are significantly shortened for stiffened tumor cells. Furthermore, we have identified PIEZO1 as the predominantly expressed mechanosensitive ion channel among the examined candidates in NK cells. Perturbation of PIEZO1 abolishes stiffness-dependent NK-cell responsiveness, significantly impairs the killing efficiency of NK cells in 3D, and substantially reduces NK-cell infiltration into 3D collagen matrices. Conversely, PIEZO1 activation enhances NK killing efficiency as well as infiltration. In conclusion, our findings demonstrate that PIEZO1-mediated mechanosensing is crucial for NK killing functions, highlighting the role of mechanosensing in NK-cell killing efficiency under 3D physiological conditions and the influence of environmental physical cues on NK-cell functions., (© 2024 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.)

Nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) is a Hodgkin lymphoma expressing functional B-cell receptors (BCR). Recently, we described a dual stimulation model of IgD+ lymphocyte-predominant cells by Moraxella catarrhalis antigen RpoC and its superantigen MID/hag, associated with extralong CDR3 and HLA-DRB1*04 or HLADRB1* 07 haplotype. The aim of the present study was to extend the antigen screening to further bacteria and viruses. The fragment antibody-binding (Fab) regions of seven new and 15 previously reported cases were analyzed. The reactivity of non-Moraxella spp.-reactive Fab regions against lysates of Rothia mucilaginosa was observed in 5/22 (22.7%) cases. Galactofuranosyl transferase (Gltf) and 2,3-butanediol dehydrogenase (Bdh) of R. mucilaginosa were identified by comparative silver- and immuno-staining in two-dimensional gels, with subsequent mass spectrometry and validation by western blots and enzyme-linked immunosorbent assay. Both R. mucilaginosa Gltf and Bdh induced BCR pathway activation and proliferation in vitro. Apoptosis was induced by recombinant Gltf/ETA'-immunotoxin conjugates in DEV cells expressing recombinant R. mucilaginosa-reactive BCR. Reactivity against M. catarrhalis RpoC was confirmed in 3/7 newly expressed BCR (total 10/22 reactive to Moraxella spp.), resulting in 15/22 (68.2%) cases with BCR reactivity against defined bacterial antigens. These findings strengthen the hypothesis of bacterial trigger contributing to subsets of NLPHL.

2-aminoethoxydiphenyl borate (2-APB) is commonly used as a tool to modulate calcium signaling in physiological studies. 2-APB has a complex pharmacology and acts as activator or inhibitor of a variety of Ca 2+ channels and transporters. While unspecific, 2-APB is one of the most-used agents to modulate store-operated calcium entry (SOCE) mediated by the STIM-gated Orai channels. Due to its boron core structure, 2-APB tends to readily hydrolyze in aqueous environment, a property that results in a complex physicochemical behavior. Here, we quantified the degree of hydrolysis in physiological conditions and identified the hydrolysis products diphenylborinic acid and 2-aminoethanol by NMR. Notably, we detected a high sensitivity of 2-APB/diphenylborinic acid towards decomposition by hydrogen peroxide to compounds such as phenylboronic acid, phenol, and boric acid, which were, in contrast to 2-APB itself and diphenylborinic acid, insufficient to affect SOCE in physiological experiments. Consequently, the efficacy of 2-APB as a Ca 2+ signal modulator strongly depends on the reactive oxygen species (ROS) production within the experimental system. The antioxidant behavior of 2-APB towards ROS and its resulting decomposition are inversely correlated to its potency to modulate Ca 2+ signaling as shown by electron spin resonance spectroscopy (ESR) and Ca 2+ imaging. Finally, we observed a strong inhibitory effect of 2-APB, i.e., its hydrolysis product diphenylborinic acid, on NADPH oxidase (NOX2) activity in human monocytes. These new 2-APB properties are highly relevant for Ca 2+ and redox signaling studies and for pharmacological application of 2-APB and related boron compounds., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Cytotoxic CD8 + T lymphocytes (CTL) eliminate infected cells or transformed tumor cells by releasing perforin-containing cytotoxic granules at the immunological synapse. The secretion of such granules depends on Ca 2+ -influx through store operated Ca 2+ channels, formed by STIM (stromal interaction molecule)-activated Orai proteins. Whereas molecular mechanisms of the secretion machinery are well understood, much less is known about the molecular machinery that regulates the efficiency of Ca 2+ -dependent target cell killing. CTL killing efficiency is of high interest considering the number of studies on CD8 + T lymphocytes modified for clinical use. Here, we isolated total RNA from primary human cells: natural killer (NK) cells, non-stimulated CD8 + T-cells, and from Staphylococcus aureus enterotoxin A (SEA) stimulated CD8 + T-cells (SEA-CTL) and conducted whole genome expression profiling by microarray experiments. Based on differential expression analysis of the transcriptome data and analysis of master regulator genes, we identified 31 candidates which potentially regulate Ca 2+ -homeostasis in CTL. To investigate a putative function of these candidates in CTL cytotoxicity, we transfected either SEA-stimulated CTL (SEA-CTL) or antigen specific CD8 + T-cell clones (CTL-MART-1) with siRNAs specific against the identified candidates and analyzed the killing capacity using a real-time killing assay. In addition, we complemented the analysis by studying the effect of inhibitory substances acting on the candidate proteins if available. Finally, to unmask their involvement in Ca 2+ dependent cytotoxicity, candidates were also analyzed under Ca 2+ -limiting conditions. Overall, we identified four hits, CCR5 (C-C chemokine receptor type five), KCNN4 (potassium calcium-activated channel subfamily N), RCAN3 (regulator of calcineurin) and BCL (B-cell lymphoma) 2 which clearly affect the efficiency of Ca 2+ dependent cytotoxicity in CTL-MART-1 cells, CCR5, BCL2, and KCNN4 in a positive manner, and RCAN3 in a negative way., Competing Interests: Declaration of Competing Interest The authors declare no competing or conflicting interests., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Despite recent advances in the therapy of diffuse large B-cell lymphoma, not otherwise specified (DLBCL), around 30% of patients develop refractory disease or relapse after first-line treatment. Recently, Ars2 was reported as the auto-antigenic target of the B-cell receptor (BCR) in approximately 25% of activated B-cell DLBCL cases. Ars2 could be used to specifically target B cells expressing Ars2-reactive BCRs. However, the optimal therapeutic format to integrate Ars2 into has yet to be determined. To mimic therapeutic antibody formats, Ars2-containing bispecific and IgG1-like constructs (BCR antigens for reverse [BAR]-bodies) were developed. Two bispecific BAR-bodies connecting single-chain antibodies against CD16 or CD3 to the BCR-binding epitope of Ars2 were constructed. Both constructs showed strong binding to U2932 cells and induced effector cell-dependent and selective cytotoxicity against U2932 cells of up to 44% at concentrations of 20 μg/ml. Additionally, IgG1-format Ars2 BAR-bodies were constructed by replacing the variable heavy- and light-chain regions of a full-length antibody with the Ars2 epitope. IgG1-format Ars2 BAR-bodies also bound selectively to U2932 and OCI-Ly3 cells and induced selective cytotoxicity of up to 60% at 10 μg/ml. In conclusion, Ars2-containing bispecific and IgG1-format BAR-bodies both are new therapeutic formats to target DLBCL cells., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2022 The Authors. eJHaem published by British Society for Haematology and John Wiley & Sons Ltd.)

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. KEY POINTS: Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells eliminate cancer cells. Both CTL and NK cells attack the same targets, but most studies address them individually. In a sequential cytotoxicity model, the interdependence of antigen-specific CTL and NK cell cytotoxicity against melanoma is quantified. High numbers of antigen-specific CTL and NK cells eliminate all melanoma cells. However, lower numbers induce resistance if secondary CTL or NK cell exposure follows initial CTL exposure or if secondary NK cell exposure follows initial NK cell exposure. On the contrary, if secondary CTL exposure follows initial NK cell exposure, cytotoxicity is enhanced. Alterations in human leucocyte antigen class I expression and interleukin-6 levels are correlated with resistance to NK cells, whereas a reduction in antigen expression is correlated with reduced CTL cytotoxicity; CTL cytotoxicity is rescued beyond control levels by exogenous antigen. This assay and the results on interdependencies will help us to understand and optimize immune therapies against cancer., (© 2022 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

A variety of intrinsic and extrinsic factors contribute to the altered efficiency of CTLs in elderly organisms. In particular, the efficacy of antiviral CD8 + T cells responses in the elderly has come back into focus since the COVID-19 pandemic outbreak. However, the exact molecular mechanisms leading to alterations in T cell function and the origin of the observed impairments have not been fully explored. Therefore, we investigated whether intrinsic changes affect the cytotoxic ability of CD8 + T cells in aging. We focused on the different subpopulations and time-resolved quantification of cytotoxicity during tumor cell elimination. We report a surprising result: Killing kinetics of CD8 + T cells from elderly mice are much faster than those of CD8 + T cells from adult mice. This is true not only in the total CD8 + T cell population but also for their effector (T EM ) and central memory (T CM ) T cell subpopulations. TIRF experiments reveal that CD8 + T cells from elderly mice possess comparable numbers of fusion events per cell, but significantly increased numbers of cells with granule fusion. Analysis of the cytotoxic granule (CG) content shows significantly increased perforin and granzyme levels and turns CD8 + T cells of elderly mice into very efficient killers. This highlights the importance of distinguishing between cell-intrinsic alterations and microenvironmental changes in elderly individuals. Our results also stress the importance of analyzing the dynamics of CTL cytotoxicity against cancer cells because, with a simple endpoint lysis analysis, cytotoxic differences could have easily been overlooked., (© 2022 The Authors. Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.)

Burkitt lymphoma (BL) represents the most aggressive B-cell-lymphoma. Beside the hallmark of IG-MYC -translocation, surface B-cell receptor (BCR) is expressed, and mutations in the BCR pathway are frequent. Coincidental infections in endemic BL, and specific extra-nodal sites suggest antigenic triggers. To explore this hypothesis, BCRs of BL cell lines and cases were screened for reactivities against a panel of bacterial lysates, lysates of Plasmodium falciparum , a custom-made virome array and against self-antigens, including post-translationally modified antigens. An atypically modified, SUMOylated isoform of Bystin, that is, SUMO1-BYSL was identified as the antigen of the BCR of cell line CA46. SUMO1-BYSL was exclusively expressed in CA46 cells with K139 as site of the SUMOylation. Secondly, an atypically acetylated isoform of HSP40 was identified as the antigen of the BCR of cell line BL41. K104 and K179 were the sites of immunogenic acetylation, and the acetylated HSP40 isoform was solely present in BL41 cells. Functionally, addition of SUMO1-BYSL and acetylated HSP40 induced BCR pathway activation in CA46 and BL41 cells, respectively. Accordingly, SUMO1-BYSL-ETA' immunotoxin, produced by a two-step intein-based conjugation, led to the specific killing of CA46 cells. Autoantibodies directed against SUMO1-BYSL were found in 3 of 14 (21.4%), and autoantibodies against acetylated HSP40 in 1/14(7.1%) patients with sporadic Burkitt-lymphoma. No reactivities against antigens of the infectious agent spectrum could be observed. These results indicate a pathogenic role of autoreactivity evoked by immunogenic post-translational modifications in a subgroup of sporadic BL including two EBV-negative BL cell lines., Competing Interests: The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported., (© 2022 The Authors. eJHaem published by British Society for Haematology and John Wiley & Sons Ltd.)

Immunological memory is important to protect humans against recurring diseases. Memory CD8 + T cells are required for quick expansion into effector cells but also provide immediate cytotoxicity against their targets. Whereas many functions of the two main cytotoxic subtypes, effector memory CD8 + T cells (T EM ) and central memory CD8 + T cells (T CM ), are well defined, single T EM and T CM cell cytotoxicity has not been quantified. To quantify cytotoxic efficiency of T EM and T CM , we developed a FRET-based single cell fluorescent assay with NALM6 target cells which allows analysis of target cell apoptosis, secondary necrosis following apoptosis, and primary necrosis after T EM - or T CM -target cell contact. Both, single cell and population cytotoxicity assays reveal a higher cytotoxic efficiency of T EM compared to T CM , as quantified by target cell apoptosis and secondary necrosis. Perforin, granzyme B, FasL, but not TRAIL expression are higher in T EM compared to T CM . Higher perforin levels (likely in combination with higher granzyme levels) mediate higher cytotoxic efficiency of T EM compared to T CM . Both, T EM and T CM need the same time to find their targets, however contact time between CTL and target, time to induce apoptosis, and time to induce secondary necrosis are all shorter for T EM . In addition, immune synapse formation in T EM appears to be slightly more efficient than in T CM . Defining and quantifying single T EM and T CM cytotoxicity and the respective mechanisms is important to optimize future subset-based immune therapies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Knörck, Schäfer, Alansary, Richter, Thurner, Hoth and Schwarz.)

TNF-related apoptosis inducing ligand (TRAIL) is expressed on cytotoxic T lymphocytes (CTLs) and TRAIL is linked to progression of diabetes. However, the impact of high glucose on TRAIL expression and its related killing function in CTLs still remains largely elusive. Here, we report that TRAIL is substantially up-regulated in CTLs in environments with high glucose (HG) both in vitro and in vivo . Non-mitochondrial reactive oxygen species, NFκB and PI3K/Akt are essential in HG-induced TRAIL upregulation in CTLs. TRAIL high CTLs induce apoptosis of pancreatic beta cell line 1.4E7. Treatment with metformin and vitamin D reduces HG-enhanced expression of TRAIL in CTLs and coherently protects 1.4E7 cells from TRAIL-mediated apoptosis. Our work suggests that HG-induced TRAIL high CTLs might contribute to the destruction of pancreatic beta cells in a hyperglycemia condition., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Yang, Denger, Diener, Küppers, Soriano-Baguet, Schäfer, Yanamandra, Zhao, Knörck, Schwarz, Hart, Lammert, Roma, Brenner, Christidis, Helms, Meese, Hoth and Qu.)

Background: The addition of rituximab to chemotherapy has substantially improved outcomes for patients with B-cell malignancies. The mechanisms of action of rituximab include activation of natural killer cells. Killer-cell immunoglobulin-like receptors (KIRs) mediate natural killer cell function through interaction with HLA. We evaluated the clinical impact of KIR-HLA genotypes on rituximab-containing therapy., Methods: For this post-hoc analysis, we used data from the RICOVER-60 trial (NCT00052936) as the discovery cohort and the CLL8 trial (NCT00281918) as the validation cohort. RICOVER-60 included patients aged 61-80 years with aggressive B-cell lymphoma treated with CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) with or without rituximab. CLL8 included patients aged 30-81 years with chronic lymphocytic leukaemia treated with chemotherapy (fludarabine and cyclophosphamide; FC) with or without rituximab. We evaluated the KIR and HLA-C status of 519 patients with available blood samples in the RICOVER-60 trial and the KIR2DS1 and HLA-C status of 549 patients with available blood samples in the CLL8 trial, and evaluated their associations with event-free survival (RICOVER-60), progression-free survival, and overall survival (RICOVER-60 and CLL8)., Findings: In the RICOVER-60 trial, 201 (39%) patients were positive for KIR2DS1, 79 (15%) were homozygous for HLA-C2, and 36 (7%) were positive for KIR2DS1 and homozygous for HLA-C2. In the CLL8 trial, 206 (38%) patients were positive for KIR2DS1, 75 (14%) were homozygous for HLA-C2, and 26 (5%) were positive for KIR2DS1 and homozygous for HLA-C2. In the RICOVER-60 trial, both KIR2DS1 and HLA-C status were identified as independent risk factors for survival. KIR2DS1 positivity, homozygosity for HLA-C2, and subsequent KIR2DS1-HLA-C status were associated with adverse clinical outcome in patients receiving rituximab-containing therapy (event-free survival for patients with KIR2DS1-HLA-C2/C2 vs all other patients, HR 2·6 [95% CI 1·4-4·7], p=0·0015; progression-free survival, 2·7 [1·5-5·1], p=0·0013; overall survival, 2·8 [1·5-5·4], p=0·0016) but not in patients receiving CHOP chemotherapy only (event-free survival, 0·9 [0·5-1·7], p=0·85; progression-free survival, 1·1 [0·6-2·0], p=0·81; overall survival, 1·2 [0·6-2·4], p=0·53). A significant interaction between KIR2DS1-HLA-C status and rituximab was observed (p=0·018 for event-free survival and p=0·034 for progression-free survival). In contrast to all other patients, those positive for KIR2DS1 and homozygous for HLA-C2 did not benefit from adding rituximab to CHOP chemotherapy (event-free survival, 1·9 [0·8-4·6], p=0·16; progression-free survival, 1·4 [0·6-3·4], p=0·48; overall survival, 1·6 [0·6-4·3], p=0·33). In the CLL8 trial, KIR2DS1-HLA-C status was confirmed as a predictive marker for benefit from rituximab therapy (p=0·024 for the interaction of KIR2DS1-HLA-C status and rituximab regarding progression-free survival). In contrast to all other patients, those positive for KIR2DS1 and homozygous for HLA-C2 did not benefit from adding rituximab to FC chemotherapy (progression-free survival, 2·1 [0·9-4·9], p=0·094; overall survival, 2·6 [0·5-12·7], p=0·21)., Interpretation: Assessment of KIR2DS1 and HLA-C genotype might identify patients who would not benefit from rituximab, thereby allowing alternative therapies to be given. Further validation of these findings in prospective clinical trials is needed., Funding: F Hoffman La Roche., Competing Interests: Declaration of interests DK-M reports personal fees, grants, and non-financial support from Novartis, ViiV Healthcare, and Gilead. SS reports personal fees, grants, and non-financial support from AbbVie, Amgen, AstraZeneca, BeiGene, Bristol Myers Squibb, Celgene, Gilead, GlaxoSmithKline, Roche, Janssen, Novartis, and Sunesis. KF reports personal fees from AbbVie and Roche, outside the submitted work. VP reports non-financial support from Abbvie, Amgen, Bristol Myers Squibb, and Roche. JTB reports personal fees from Merck Sharp & Dohme and Incyte Pharmaceutical, and non-financial support from Gilead. LTh reports grants from the non-profit Wilhelm Sander Foundation, personal fees from EUSA Pharma and AstraZeneca, and non-financial support from AbbVie, EUSA Pharma, and Janssen. MB reports grants from the non-profit Deutsche José-Carreras Leukaemie Stiftung. All other authors declare no competing interests., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Visualizing interactions between cells and the extracellular matrix (ECM) mesh is important to understand cell behavior and regulatory mechanisms by the extracellular environment. However, long term visualization of three-dimensional (3D) matrix structures remains challenging mainly due to photobleaching or blind spots perpendicular to the imaging plane. Here, we combine label-free light-sheet scattering microcopy (LSSM) and fluorescence microscopy to solve these problems. We verified that LSSM can reliably visualize structures of collagen matrices from different origin including bovine, human and rat tail. The quality and intensity of collagen structure images acquired by LSSM did not decline with time. LSSM offers abundant wavelength choice to visualize matrix structures, maximizing combination possibilities with fluorescently-labelled cells, allowing visualizing of long-term ECM-cell interactions in 3D. Interestingly, we observed ultrathin thread-like structures between cells and matrix using LSSM, which were not observed by normal fluorescence microscopy. Transient local alignment of matrix by cell-applied forces can be observed. In summary, LSSM provides a powerful and robust approach to investigate the complex interplay between cells and ECM., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Zhou, Zhao, Yanamandra, Hoth and Qu.)

Background: Barth syndrome (BTHS) is caused by mutations of the gene encoding tafazzin, which catalyzes maturation of mitochondrial cardiolipin and often manifests with systolic dysfunction during early infancy. Beyond the first months of life, BTHS cardiomyopathy typically transitions to a phenotype of diastolic dysfunction with preserved ejection fraction, blunted contractile reserve during exercise, and arrhythmic vulnerability. Previous studies traced BTHS cardiomyopathy to mitochondrial formation of reactive oxygen species (ROS). Because mitochondrial function and ROS formation are regulated by excitation-contraction coupling, integrated analysis of mechano-energetic coupling is required to delineate the pathomechanisms of BTHS cardiomyopathy., Methods: We analyzed cardiac function and structure in a mouse model with global knockdown of tafazzin ( Taz -KD) compared with wild-type littermates. Respiratory chain assembly and function, ROS emission, and Ca 2+ uptake were determined in isolated mitochondria. Excitation-contraction coupling was integrated with mitochondrial redox state, ROS, and Ca 2+ uptake in isolated, unloaded or preloaded cardiac myocytes, and cardiac hemodynamics analyzed in vivo., Results: Taz -KD mice develop heart failure with preserved ejection fraction (>50%) and age-dependent progression of diastolic dysfunction in the absence of fibrosis. Increased myofilament Ca 2+ affinity and slowed cross-bridge cycling caused diastolic dysfunction, in part, compensated by accelerated diastolic Ca 2+ decay through preactivated sarcoplasmic reticulum Ca 2 + -ATPase. Taz deficiency provoked heart-specific loss of mitochondrial Ca 2+ uniporter protein that prevented Ca 2+ -induced activation of the Krebs cycle during β-adrenergic stimulation, oxidizing pyridine nucleotides and triggering arrhythmias in cardiac myocytes. In vivo, Taz -KD mice displayed prolonged QRS duration as a substrate for arrhythmias, and a lack of inotropic response to β-adrenergic stimulation. Cellular arrhythmias and QRS prolongation, but not the defective inotropic reserve, were restored by inhibiting Ca 2+ export through the mitochondrial Na + /Ca 2+ exchanger. All alterations occurred in the absence of excess mitochondrial ROS in vitro or in vivo., Conclusions: Downregulation of mitochondrial Ca 2+ uniporter, increased myofilament Ca 2+ affinity, and preactivated sarcoplasmic reticulum Ca 2+ -ATPase provoke mechano-energetic uncoupling that explains diastolic dysfunction and the lack of inotropic reserve in BTHS cardiomyopathy. Furthermore, defective mitochondrial Ca 2+ uptake provides a trigger and a substrate for ventricular arrhythmias. These insights can guide the ongoing search for a cure of this orphaned disease.

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 PI 3 K/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., (©2021 American Association for Cancer Research.)

Efficacy of cytotoxic T lymphocyte (CTL)-based immunotherapy is still unsatisfactory against solid tumors, which are frequently characterized by condensed extracellular matrix. Here, using a unique 3D killing assay, we identify that the killing efficiency of primary human CTLs is substantially impaired in dense collagen matrices. Although the expression of cytotoxic proteins in CTLs remained intact in dense collagen, CTL motility was largely compromised. Using light-sheet microscopy, we found that persistence and velocity of CTL migration was influenced by the stiffness and porosity of the 3D matrix. Notably, 3D CTL velocity was strongly correlated with their nuclear deformability, which was enhanced by disruption of the microtubule network especially in dense matrices. Concomitantly, CTL migration, search efficiency, and killing efficiency in dense collagen were significantly increased in microtubule-perturbed CTLs. In addition, the chemotherapeutically used microtubule inhibitor vinblastine drastically enhanced CTL killing efficiency in dense collagen. Together, our findings suggest targeting the microtubule network as a promising strategy to enhance efficacy of CTL-based immunotherapy against solid tumors, especially stiff solid tumors., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Zhao, Zhou, Khan, Alansary, Friedmann, Yang, Schwarz, del Campo, Hoth and Qu.)

Mantle cell lymphoma (MCL) accounts for 5%-10% of all lymphomas. The disease's genetic hallmark is the t(11; 14)(q13; q32) translocation. In younger patients, the first-line treatment is chemoimmunotherapy followed by autologous stem cell transplantation. Upon disease progression, novel and targeted agents such as the BTK inhibitor ibrutinib, the BCL-2 inhibitor venetoclax, or the combination of both are increasingly used, but even after allogeneic stem cell transplantation or CAR T-cell therapy, MCL remains incurable for most patients. Chronic antigenic stimulation of the B-cell receptor (BCR) is thought to be essential for the pathogenesis of many B-cell lymphomas. LRPAP1 has been identified as the autoantigenic BCR target in about 1/3 of all MCLs. Thus, LRPAP1 could be used to target MCL cells, however, there is currently no optimal therapeutic format to integrate LRPAP1. We have therefore integrated LRPAP1 into a concept termed BAR, for B-cell receptor antigens for reverse targeting. A bispecific BAR body was synthesized consisting of the lymphoma-BCR binding epitope of LRPAP1 and a single chain fragment targeting CD3 or CD16 to recruit/engage T or NK cells. In addition, a BAR body consisting of an IgG1 antibody and the lymphoma-BCR binding epitope of LRPAP1 replacing the variable regions was synthesized. Both BAR bodies mediated highly specific cytotoxic effects against MCL cells in a dose-dependent manner at 1-20 µg/mL. In conclusion, LRPAP1 can substitute variable antibody regions in different formats to function in a new therapeutic approach to treat MCL., (Copyright © 2021 the Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the European Hematology Association.)

Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells are required for host defense. They destroy malignant target cells like cancer cells. Among metal cations, Ca 2+ plays a prescinded role for CTL and NK cytotoxicity as it is the only cation used as ubiquitous second messenger. Measuring intracellular Ca 2+ concentrations [Ca 2+ ] int in single cells has greatly changed our understanding of Ca 2+ signaling. Yet, comparing the role of Ca 2+ in the pre-[Ca 2+ ] int and [Ca 2+ ] int measurement era reveals that even in the pre-[Ca 2+ ] int measurement era (before 1980), the functions of Ca 2+ and some other metal cations for the cytotoxic immune response were well established. It was even shown that Ca 2+ influx across the plasma membrane but not Ca 2+ release from intracellular sources is relevant for lymphocyte cytotoxicity and that very little Ca 2+ is needed for efficient lymphocyte cytotoxicity against cancer cells. In the [Ca 2+ ] int measurement era after 1980, many of the important findings were better and more quantitatively refined and in addition the molecules important for Ca 2+ transport were defined. The unexpected finding that there is a Ca 2+ optimum of CTL and NK cell cytotoxicity deserves some attention and may be important for anti-cancer therapy., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Progress in our understanding of mechanotransduction events requires noninvasive methods for the manipulation of forces at molecular scale in physiological environments. Inspired by cellular mechanisms for force application (i.e. motor proteins pulling on cytoskeletal fibers), we present a unique molecular machine that can apply forces at cell-matrix and cell-cell junctions using light as an energy source. The key actuator is a light-driven rotatory molecular motor linked to polymer chains, which is intercalated between a membrane receptor and an engineered biointerface. The light-driven actuation of the molecular motor is converted in mechanical twisting of the entangled polymer chains, which will in turn effectively "pull" on engaged cell membrane receptors (e.g., integrins, T cell receptors) within the illuminated area. Applied forces have physiologically-relevant magnitude and occur at time scales within the relevant ranges for mechanotransduction at cell-friendly exposure conditions, as demonstrated in force-dependent focal adhesion maturation and T cell activation experiments. Our results reveal the potential of nanomotors for the manipulation of living cells at the molecular scale and demonstrate a functionality which at the moment cannot be achieved by other technologies for force application.

Chronic obstructive lung disease (COPD) and lung cancer are both caused by smoking and often occur as comorbidity. The programmed cell death protein 1/programmed cell death ligand 1 (PD-1/PD-L1) axis is an important canonic immunoregulatory pathway, and antibodies that specifically block PD-1 or PD-L1 have demonstrated efficacy as therapeutic agents for non-small cell lung cancer. The role of the PD-1/PD-L1 axis in the pathogenesis of COPD is unknown. Here, we analyzed the function of the PD-1/PD-L1 axis in preclinical COPD models and evaluated the concentrations of PD-1 and PD-L1 in human serum and bronchoalveolar lavage (BAL) fluids as biomarkers for COPD. Anti-PD-1 treatment decreased lung damage and neutrophilic inflammation in mice chronically exposed to cigarette smoke (CS) or nontypeable Haemophilus influenzae (NTHi). Ex vivo stimulated macrophages obtained from anti-PD-1-treated mice released reduced amounts of inflammatory cytokines. PD-L1 concentrations correlated positively with PD-1 concentrations in human serum and BAL fluids. Lung sections obtained from patients with COPD stained positive for PD-L1. Our data indicate that the PD-1/PD-L1 axis is involved in developing inflammation and tissue destruction in COPD. Inflammation-induced activation of the PD-1 pathway may contribute to disease progression.

The analysis of T cell responses to mechanical properties of antigen presenting cells (APC) is experimentally challenging at T cell-APC interfaces. Soft hydrogels with adjustable mechanical properties and biofunctionalization are useful reductionist models to address this problem. Here, we report a methodology to fabricate micropatterned soft hydrogels with defined stiffness to form spatially confined T cell/hydrogel contact interfaces at micrometer scale. Using automatized microcontact printing we prepared arrays of anti-CD3 microdots on poly(acrylamide) hydrogels with Young's Modulus in the range of 2 to 50 kPa. We optimized the printing process to obtain anti-CD3 microdots with constant area (50 µm 2 , corresponding to 8 µm diameter) and comparable anti-CD3 density on hydrogels of different stiffness. The anti-CD3 arrays were recognized by T cells and restricted cell attachment to the printed areas. To test functionality of the hydrogel-T cell contact, we analyzed several key events downstream of T cell receptor (TCR) activation. Anti-CD3 arrays on hydrogels activated calcium influx, induced rearrangement of the actin cytoskeleton, and led to Zeta-chain-associated protein kinase 70 (ZAP70) phosphorylation. Interestingly, upon increase in the stiffness, ZAP70 phosphorylation was enhanced, whereas the rearrangements of F-actin (F-actin clearance) and phosphorylated ZAP70 (ZAP70/pY centralization) were unaffected. Our results show that micropatterned hydrogels allow tuning of stiffness and receptor presentation to analyze TCR mediated T cell activation as function of mechanical, biochemical, and geometrical parameters., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier Ltd.)

The B-cell receptor (BCR) signaling pathway is a crucial pathway of B cells, both for their survival and for antigen-mediated activation, proliferation and differentiation. Its activation is also critical for the genesis of many lymphoma types. BCR-mediated lymphoma proliferation may be caused by activating BCR-pathway mutations and/or by active or tonic stimulation of the BCR. BCRs of lymphomas have frequently been described as polyreactive. In this review, the role of specific target antigens of the BCRs of lymphomas is highlighted. These antigens have been found to be restricted to specific lymphoma entities. The antigens can be of infectious origin, such as H. pylori in gastric MALT lymphoma or RpoC of M. catarrhalis in nodular lymphocyte predominant Hodgkin lymphoma, or they are autoantigens. Examples of such autoantigens are the BCR itself in chronic lymphocytic leukemia, LRPAP1 in mantle cell lymphoma, hyper-N-glycosylated SAMD14/neurabin-I in primary central nervous system lymphoma, hypo-phosphorylated ARS2 in diffuse large B-cell lymphoma, and hyper-phosphorylated SLP2, sumoylated HSP90 or saposin C in plasma cell dyscrasia. Notably, atypical posttranslational modifications are often responsible for the immunogenicity of many autoantigens. Possible therapeutic approaches evolving from these specific antigens are discussed., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Thurner, Hartmann, Neumann, Hoth, Stilgenbauer, Küppers, Preuss and Bewarder.)

Recently, neurabin-I and SAMD14 have been described as the autoantigenic target of approximately 66% of B-cell receptors (BCRs) of primary central nervous system lymphomas (PCNSL). Neurabin-I and SAMD14 share a highly homologous SAM domain that becomes immunogenic after atypical hyper-N-glycosylation (SAMD14 at ASN339 and neurabin-I at ASN1277). This post-translational modification of neurabin-I and SAMD14 seems to lead to a chronic immune reaction with B-cell receptor activation contributing to lymphoma genesis of PCNSLs. The selective tropism of PCNSL to the CNS corresponds well to the neurabin-I and SAMD14 protein expression pattern. When conjugated to Pseudomonas Exotoxin A (ETA´), the PCNSL reactive epitope exerts cytotoxic effects on lymphoma cells expressing a SAMD14/neurabin-I reactive BCR. Thus, the reactive epitopes of SAMD14/neurabin-I might be useful to establish additional therapeutic strategies against PCNSL. To test this possibility, we integrated the PCNSL-reactive epitope of SAMD14/neurabin-I into a heavy-chain-only Fab antibody format in substitution of the variable region. Specific binding of the prokaryotically produced SAMD14/neurabin-I Fab-antibody to lymphoma cells and their internalization were determined by flow cytometry. Since no established EBV-negative PCNSL cell line exists, we used the ABC-DLBCL cell lines OCI-Ly3 and U2932, which were transfected to express a SAMD14/neurabin-I reactive BCR. The SAMD14/neurabin-I Fab antibody bound specifically to DLBCL cells expressing a BCR with reactivity to SAMD14/neurabin-I and not to unmanipulated DLBCL cell lines. Eukaryotically produced full-length IgG antibodies are well established as immunotherapy format. Therefore, the PCNSL-reactive epitope of SAMD14/neurabin-I was cloned into a full-length IgG1 format replacing the variable domains of the light and heavy chains. The IgG1-format SAMD14/neurabin-I construct was found to specifically bind to target lymphoma cells expressing a SAMD14/neurabin-I reactive B cell receptor. In addition, it induced dose-dependent relative cytotoxicity against these lymphoma cells when incubated with PBMCs. Control DLBCL cells are not affected at any tested concentration. When integrated into the Fab-format and IgG1-format, the PCNSL-reactive epitope of SAMD14/neurabin-I functions as B -cell receptor A ntigen for R everse targeting (BAR). In particular, the IgG1-format BAR-body approach represents a very attractive therapeutic format for the treatment of PCNSLs, considering its specificity against SAMD14/neurabin-I reactive BCRs and the well-known pharmacodynamic properties of IgG antibodies., (Copyright © 2020 Bewarder, Kiefer, Moelle, Goerens, Stilgenbauer, Christofyllakis, Kaddu-Mulindwa, Fadle, Regitz, Neumann, Hoth, Preuss, Pfreundschuh and Thurner.)

Store-operated calcium entry (SOCE) through STIM-gated ORAI channels governs vital cellular functions. In this context, SOCE controls cellular redox signaling and is itself regulated by redox modifications. However, the molecular mechanisms underlying this calcium-redox interplay and the functional outcomes are not fully understood. Here, we examine the role of STIM2 in SOCE redox regulation. Redox proteomics identify cysteine 313 as the main redox sensor of STIM2 in vitro and in vivo. Oxidative stress suppresses SOCE and calcium currents in cells overexpressing STIM2 and ORAI1, an effect that is abolished by mutation of cysteine 313. FLIM and FRET microscopy, together with MD simulations, indicate that oxidative modifications of cysteine 313 alter STIM2 activation dynamics and thereby hinder STIM2-mediated gating of ORAI1. In summary, this study establishes STIM2-controlled redox regulation of SOCE as a mechanism that affects several calcium-regulated physiological processes, as well as stress-induced pathologies., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Key Points: Mitochondrial Ca 2+ uptake stimulates the Krebs cycle to regenerate the reduced forms of pyridine nucleotides (NADH, NADPH and FADH 2 ) required for ATP production and reactive oxygen species (ROS) elimination. Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) has been proposed to regulate mitochondrial Ca 2+ uptake via mitochondrial Ca 2+ uniporter phosphorylation. We used two mouse models with either global deletion of CaMKIIδ (CaMKIIδ knockout) or cardiomyocyte-specific deletion of CaMKIIδ and γ (CaMKIIδ/γ double knockout) to interrogate whether CaMKII controls mitochondrial Ca 2+ uptake in isolated mitochondria and during β-adrenergic stimulation in cardiac myocytes. CaMKIIδ/γ did not control Ca 2+ uptake, respiration or ROS emission in isolated cardiac mitochondria, nor in isolated cardiac myocytes, during β-adrenergic stimulation and pacing. The results of the present study do not support a relevant role of CaMKII for mitochondrial Ca 2+ uptake in cardiac myocytes under physiological conditions., Abstract: Mitochondria are the main source of ATP and reactive oxygen species (ROS) in cardiac myocytes. Furthermore, activation of the mitochondrial permeability transition pore (mPTP) induces programmed cell death. These processes are essentially controlled by Ca 2+ , which is taken up into mitochondria via the mitochondrial Ca 2+ uniporter (MCU). It was recently proposed that Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) regulates Ca 2+ uptake by interacting with the MCU, thereby affecting mPTP activation and programmed cell death. In the present study, we investigated the role of CaMKII under physiological conditions in which mitochondrial Ca 2+ uptake matches energy supply to the demand of cardiac myocytes. Accordingly, we measured mitochondrial Ca 2+ uptake in isolated mitochondria and cardiac myocytes harvested from cardiomyocyte-specific CaMKII δ and γ double knockout (KO) (CaMKIIδ/γ DKO) and global CaMKIIδ KO mice. To simulate a physiological workload increase, cardiac myocytes were subjected to β-adrenergic stimulation (by isoproterenol superfusion) and an increase in stimulation frequency (from 0.5 to 5 Hz). No differences in mitochondrial Ca 2+ accumulation were detected in isolated mitochondria or cardiac myocytes from both CaMKII KO models compared to wild-type littermates. Mitochondrial redox state and ROS production were unchanged in CaMKIIδ/γ DKO, whereas we observed a mild oxidation of mitochondrial redox state and an increase in H 2 O 2 emission from CaMKIIδ KO cardiac myocytes exposed to an increase in workload. In conclusion, the results obtained in the present study do not support the regulation of mitochondrial Ca 2+ uptake via the MCU or mPTP activation by CaMKII in cardiac myocytes under physiological conditions., (© 2019 The Authors. The Journal of Physiology © 2019 The Physiological Society.)

Dendritic cells "patrol" the human body to detect pathogens. In their search, dendritic cells perform a random walk by amoeboid migration. The efficiency of pathogen detection depends on the properties of the random walk. It is not known how the dendritic cells control these properties. Here, we quantify dendritic cell migration under well-defined 2-dimensional confinement and in a 3-dimensional collagen matrix through recording their long-term trajectories. We find 2 different migration states: persistent migration, during which the dendritic cells move along curved paths, and diffusive migration, which is characterized by successive sharp turns. These states exhibit differences in the actin distributions. Our theoretical and experimental analyses indicate that this kind of motion can be generated by spontaneous actin polymerization waves that contribute to dendritic cell polarization and migration. The relative distributions of persistent and diffusive migration can be changed by modification of the molecular actin filament nucleation and assembly rates. Thus, dendritic cells can control their migration patterns and adapt to specific environments. Our study offers an additional perspective on how dendritic cells tune their searches for pathogens., Competing Interests: The authors declare no competing interest.

Amplitude and kinetics of intracellular Ca 2+ signals ([Ca 2+ ] int ) determine many immune cell functions. To mimic in vivo changes of [Ca 2+ ] int in human immune cells, two approaches may be best suited: 1) Analyze primary human immune cells taken from blood under conditions resembling best physiological or pathophysiological conditions. 2.) Analyze the immune system in vivo or ex vivo in explanted tissue from small vertebrate animals, such as mice. With the help of genetically encoded Ca 2+ indicators and intravital microscopy, [Ca 2+ ] int have been investigated in murine T lymphocytes (T cells) in vivo during the last five years and in explanted lymph node (LN) during the last 10 years. There are several important reasons to compare [Ca 2+ ] int measured in primary murine T lymphocytes in vivo and in vitro with [Ca 2+ ] int measured in primary human T lymphocytes in vitro. First, how do human and murine data compare? Second, how do in vivo and in vitro data compare? Third, can in vitro data predict in vivo data? The last point is particularly important considering the many technical challenges that limit in vivo measurements and to reduce the number of animals sacrificed. This review summarizes and compares the results of the available publications on in vivo and in vitro [Ca 2+ ] int measurements in T lymphocytes stimulated focally by antigen-presenting cells (APC) after forming an immunological synapse., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Chronic obstructive pulmonary disease (COPD) is associated with neutrophilic lung inflammation and CD8 T cell exhaustion and is an important risk factor for the development of non-small cell lung cancer (NSCLC). The clinical response to programmed cell death-1 (PD-1) blockade in NSCLC patients is variable and likely affected by a coexisting COPD. The pro-inflammatory cytokine interleukin-17C (IL-17C) promotes lung inflammation and is present in human lung tumors. Here, we used a Kras-driven lung cancer model to examine the function of IL-17C in inflammation-promoted tumor growth. Genetic ablation of Il-17c resulted in a decreased recruitment of inflammatory cells into the tumor microenvironment, a decreased expression of tumor-promoting cytokines (e.g. interleukin-6 (IL-6)), and a reduced tumor proliferation in the presence of Haemophilus influenzae- (NTHi) induced COPD-like lung inflammation. Chronic COPD-like inflammation was associated with the expression of PD-1 in CD8 lymphocytes and the membrane expression of the programmed death ligand (PD-L1) independent of IL-17C. Tumor growth was decreased in Il-17c deficient mice but not in wildtype mice after anti-PD-1 treatment. Our results suggest that strategies targeting innate immune mechanisms, such as blocking of IL-17C, may improve the response to anti-PD-1 treatment in lung cancer patients.

The tumor microenvironment (TME) is shaped by cancer and noncancerous cells, the extracellular matrix, soluble factors, and blood vessels. Interactions between the cells, matrix, soluble factors, and blood vessels generate this complex heterogeneous microenvironment. The TME may be metabolically beneficial or unbeneficial for tumor growth, it may favor or not favor a productive immune response against tumor cells, or it may even favor conditions suited to hijacking the immune system for benefitting tumor growth. Soluble factors relevant for TME include oxygen, reactive oxygen species (ROS), ATP, Ca 2+ , H⁺, growth factors, or cytokines. Ca 2+ plays a prominent role in the TME because its concentration is directly linked to cancer cell proliferation, apoptosis, or migration but also to immune cell function. Stromal-interaction molecules (STIM)-activated Orai channels are major Ca 2+ entry channels in cancer cells and immune cells, they are upregulated in many tumors, and they are strongly regulated by ROS. Thus, STIM and Orai are interesting candidates to regulate cancer cell fate in the TME. In this review, we summarize the current knowledge about the function of ROS and STIM/Orai in cancer cells; discuss their interdependencies; and propose new hypotheses how TME, ROS, and Orai channels influence each other.