@article{DmochewitzFoertschZwergeretal.2013, author = {Dmochewitz, Lydia and F{\"o}rtsch, Christina and Zwerger, Christian and Vaeth, Martin and Felder, Edward and Huber-Lang, Markus and Barth, Holger}, title = {A Recombinant Fusion Toxin Based on Enzymatic Inactive C3bot1 Selectively Targets Macrophages}, series = {PLoS ONE}, volume = {8}, journal = {PLoS ONE}, number = {1}, doi = {10.1371/journal.pone.0054517}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-131189}, pages = {e54517}, year = {2013}, abstract = {Background: The C3bot1 protein (~23 kDa) from Clostridium botulinum ADP-ribosylates and thereby inactivates Rho. C3bot1 is selectively taken up into the cytosol of monocytes/macrophages but not of other cell types such as epithelial cells or fibroblasts. Most likely, the internalization occurs by a specific endocytotic pathway via acidified endosomes. Methodology/Principal Findings: Here, we tested whether enzymatic inactive C3bot1E174Q serves as a macrophage-selective transport system for delivery of enzymatic active proteins into the cytosol of such cells. Having confirmed that C3bot1E174Q does not induce macrophage activation, we used the actin ADP-ribosylating C2I (~50 kDa) from Clostridium botulinum as a reporter enzyme for C3bot1E174Q-mediated delivery into macrophages. The recombinant C3bot1E174Q-C2I fusion toxin was cloned and expressed as GST-protein in Escherichia coli. Purified C3bot1E174Q-C2I was recognized by antibodies against C2I and C3bot and showed C2I-specific enzyme activity in vitro. When applied to cultured cells C3bot1E174Q-C2I ADP-ribosylated actin in the cytosol of macrophages including J774A.1 and RAW264.7 cell lines as well as primary cultured human macrophages but not of epithelial cells. Together with confocal fluorescence microscopy experiments, the biochemical data indicate the selective uptake of a recombinant C3-fusion toxin into the cytosol of macrophages. Conclusions/Significance: In summary, we demonstrated that C3bot1E174Q can be used as a delivery system for fast, selective and specific transport of enzymes into the cytosol of living macrophages. Therefore, C3-based fusion toxins can represent valuable molecular tools in experimental macrophage pharmacology and cell biology as well as attractive candidates to develop new therapeutic approaches against macrophage-associated diseases.}, language = {en} } @article{JostKleinBrandetal.2023, author = {Jost, Priska and Klein, Franziska and Brand, Benjamin and Wahl, Vanessa and Wyatt, Amanda and Yildiz, Daniela and Boehm, Ulrich and Niemeyer, Barbara A. and Vaeth, Martin and Alansary, Dalia}, title = {Acute downregulation but not genetic ablation of murine MCU impairs suppressive capacity of regulatory CD4 T cells}, series = {International Journal of Molecular Sciences}, volume = {24}, journal = {International Journal of Molecular Sciences}, number = {9}, issn = {1422-0067}, doi = {10.3390/ijms24097772}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-313621}, year = {2023}, abstract = {By virtue of mitochondrial control of energy production, reactive oxygen species (ROS) generation, and maintenance of Ca\(^{2+}\) homeostasis, mitochondria play an essential role in modulating T cell function. The mitochondrial Ca\(^{2+}\) uniporter (MCU) is the pore-forming unit in the main protein complex mediating mitochondrial Ca\(^{2+}\) uptake. Recently, MCU has been shown to modulate Ca\(^{2+}\) signals at subcellular organellar interfaces, thus fine-tuning NFAT translocation and T cell activation. The mechanisms underlying this modulation and whether MCU has additional T cell subpopulation-specific effects remain elusive. However, mice with germline or tissue-specific ablation of Mcu did not show impaired T cell responses in vitro or in vivo, indicating that 'chronic' loss of MCU can be functionally compensated in lymphocytes. The current work aimed to specifically investigate whether and how MCU influences the suppressive potential of regulatory CD4 T cells (Treg). We show that, in contrast to genetic ablation, acute siRNA-mediated downregulation of Mcu in murine Tregs results in a significant reduction both in mitochondrial Ca\(^{2+}\) uptake and in the suppressive capacity of Tregs, while the ratios of Treg subpopulations and the expression of hallmark transcription factors were not affected. These findings suggest that permanent genetic inactivation of MCU may result in compensatory adaptive mechanisms, masking the effects on the suppressive capacity of Tregs.}, language = {en} } @article{KleinHesslingMuhammadKleinetal.2017, author = {Klein-Hessling, Stefan and Muhammad, Khalid and Klein, Matthias and Pusch, Tobias and Rudolf, Ronald and Fl{\"o}ter, Jessica and Qureischi, Musga and Beilhack, Andreas and Vaeth, Martin and Kummerow, Carsten and Backes, Christian and Schoppmeyer, Rouven and Hahn, Ulrike and Hoth, Markus and Bopp, Tobias and Berberich-Siebelt, Friederike and Patra, Amiya and Avots, Andris and M{\"u}ller, Nora and Schulze, Almut and Serfling, Edgar}, title = {NFATc1 controls the cytotoxicity of CD8\(^{+}\) T cells}, series = {Nature Communications}, volume = {8}, journal = {Nature Communications}, number = {511}, doi = {10.1038/s41467-017-00612-6}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-170353}, year = {2017}, abstract = {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.}, language = {en} } @article{MuellerQuandtMarienfeldetal.2013, author = {Mueller, Kerstin and Quandt, Jasmin and Marienfeld, Ralf B. and Weihrich, Petra and Fiedler, Katja and Claussnitzer, Melina and Laumen, Helmut and Vaeth, Martin and Berberich-Siebelt, Frederike and Serfling, Edgar and Wirth, Thomas and Brunner, Cornelia}, title = {Octamer-dependent transcription in T cells is mediated by NFAT and \(NF-\kappa B\)}, series = {Nucleic Acids Research}, volume = {41}, journal = {Nucleic Acids Research}, number = {4}, issn = {1362-4962}, doi = {10.1093/nar/gks1349}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-123280}, pages = {2138-2154}, year = {2013}, abstract = {The transcriptional co-activator BOB.1/OBF.1 was originally identified in B cells and is constitutively expressed throughout B cell development. BOB.1/OBF.1 associates with the transcription factors Oct1 and Oct2, thereby enhancing octamer-dependent transcription. In contrast, in T cells, BOB.1/OBF.1 expression is inducible by treatment of cells with PMA/Ionomycin or by antigen receptor engagement, indicating a marked difference in the regulation of BOB.1/OBF.1 expression in B versus T cells. The molecular mechanisms underlying the differential expression of BOB.1/OBF.1 in T and B cells remain largely unknown. Therefore, the present study focuses on mechanisms controlling the transcriptional regulation of BOB.1/OBF.1 and Oct2 in T cells. We show that both calcineurin- and \(NF-\kappa B\)-inhibitors efficiently attenuate the expression of BOB.1/OBF.1 and Oct2 in T cells. In silico analyses of the BOB.1/OBF.1 promoter revealed the presence of previously unappreciated combined NFAT/\(NF-\kappa B\) sites. An array of genetic and biochemical analyses illustrates the involvement of the \(Ca^{2+}\)/calmodulin-dependent phosphatase calcineurin as well as NFAT and \(NF-\kappa B\) transcription factors in the transcriptional regulation of octamer-dependent transcription in T cells. Conclusively, impaired expression of BOB.1/OBF.1 and Oct2 and therefore a hampered octamer-dependent transcription may participate in T cell-mediated immunodeficiency caused by the deletion of NFAT or \(NF-\kappa B\) transcription factors.}, language = {en} } @article{SaintFleurLominyMausVaethetal.2018, author = {Saint Fleur-Lominy, Shella and Maus, Mate and Vaeth, Martin and Lange, Ingo and Zee, Isabelle and Suh, David and Liu, Cynthia and Wu, Xiaojun and Tikhonova, Anastasia and Aifantis, Iannis and Feske, Stefan}, title = {STIM1 and STIM2 Mediate Cancer-Induced Inflammation in T Cell Acute Lymphoblastic Leukemia}, series = {Cell Reports}, volume = {24}, journal = {Cell Reports}, number = {11}, doi = {10.1016/j.celrep.2018.08.030}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-227259}, pages = {3045-3060}, year = {2018}, abstract = {T cell acute lymphoblastic leukemia (T-ALL) is commonly associated with activating mutations in the NOTCH1 pathway. Recent reports have shown a link between NOTCH1 signaling and intracellular Ca2+ homeostasis in T-ALL. Here, we investigate the role of store-operated Ca2+ entry (SOCE) mediated by the Ca2+ channel ORAI1 and its activators STIM1 and STIM2 in T-ALL. Deletion of STIM1 and STIM2 in leukemic cells abolishes SOCE and significantly prolongs the survival of mice in a NOTCH1-dependent model of T-ALL. The survival advantage is unrelated to the leukemic cell burden but is associated with the SOCE-dependent ability of malignant T lymphoblasts to cause inflammation in leukemia-infiltrated organs. Mice with STIM1/STIM2-deficient T-ALL show a markedly reduced necroinflammatory response in leukemia-infiltrated organs and downregulation of signaling pathways previously linked to cancer-induced inflammation. Our study shows that leukemic T lymphoblasts cause inflammation of leukemia-infiltrated organs that is dependent on SOCE.}, language = {en} } @misc{SerflingAvotsKleinHesslingetal.2012, author = {Serfling, Edgar and Avots, Andris and Klein-Hessling, Stefan and Rudolf, Ronald and Vaeth, Martin and Berberich-Siebelt, Friederike}, title = {NFATc1/alphaA: The other Face of NFAT Factors in Lymphocytes}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-75748}, year = {2012}, abstract = {In effector T and B cells immune receptor signals induce within minutes a rise of intracellular Ca++, the activation of the phosphatase calcineurin and the translocation of NFAT transcription factors from cytosol to nucleus. In addition to this first wave of NFAT activation, in a second step the occurrence of NFATc1/αA, a short isoform of NFATc1, is strongly induced. Upon primary stimulation of lymphocytes the induction of NFATc1/αA takes place during the G1 phase of cell cycle. Due to an auto-regulatory feedback circuit high levels of NFATc1/αA are kept constant during persistent immune receptor stimulation. Contrary to NFATc2 and further NFATc proteins which dampen lymphocyte proliferation, induce anergy and enhance activation induced cell death (AICD), NFATc1/αA supports antigenmediated proliferation and protects lymphocytes against rapid AICD. Whereas high concentrations of NFATc1/αA can also lead to apoptosis, in collaboration with NF-κB-inducing co-stimulatory signals they support the survival of mature lymphocytes in late phases after their activation. However, if dysregulated, NFATc1/αA appears to contribute to lymphoma genesis and - as we assume - to further disorders of the lymphoid system. While the molecular details of NFATc1/αA action and its contribution to lymphoid disorders have to be investigated, NFATc1/αA differs in its generation and function markedly from all the other NFAT proteins which are expressed in lymphoid cells. Therefore, it represents a prime target for causal therapies of immune disorders in future.}, subject = {Medizin}, language = {en} } @article{VaethWangEcksteinetal.2019, author = {Vaeth, Martin and Wang, Yin-Hu and Eckstein, Miriam and Yang, Jun and Silverman, Gregg J. and Lacruz, Rodrigo S. and Kannan, Kasthuri and Feske, Stefan}, title = {Tissue resident and follicular Treg cell differentiation is regulated by CRAC channels}, series = {Nature Communications}, volume = {10}, journal = {Nature Communications}, doi = {10.1038/s41467-019-08959-8}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-232148}, year = {2019}, abstract = {T regulatory (Treg) cells maintain immunological tolerance and organ homeostasis. Activated Treg cells differentiate into effector Treg subsets that acquire tissue-specific functions. Ca2+ influx via Ca2+ release-activated Ca2+ (CRAC) channels formed by STIM and ORAI proteins is required for the thymic development of Treg cells, but its function in mature Treg cells remains unclear. Here we show that deletion of Stim1 and Stim2 genes in mature Treg cells abolishes Ca2+ signaling and prevents their differentiation into follicular Treg and tissue-resident Treg cells. Transcriptional profiling of STIM1/STIM2-deficient Treg cells reveals that Ca2+ signaling regulates transcription factors and signaling pathways that control the identity and effector differentiation of Treg cells. In the absence of STIM1/STIM2 in Treg cells, mice develop a broad spectrum of autoantibodies and fatal multiorgan inflammation. Our findings establish a critical role of CRAC channels in controlling lineage identity and effector functions of Treg cells.}, language = {en} } @article{WuZhaoHochreinetal.2023, author = {Wu, Hao and Zhao, Xiufeng and Hochrein, Sophia M. and Eckstein, Miriam and Gubert, Gabriela F. and Kn{\"o}pper, Konrad and Mansilla, Ana Maria and {\"O}ner, Arman and Doucet-Ladev{\`e}ze, Remi and Schmitz, Werner and Ghesqui{\`e}re, Bart and Theurich, Sebastian and Dudek, Jan and Gasteiger, Georg and Zernecke, Alma and Kobold, Sebastian and Kastenm{\"u}ller, Wolfgang and Vaeth, Martin}, title = {Mitochondrial dysfunction promotes the transition of precursor to terminally exhausted T cells through HIF-1α-mediated glycolytic reprogramming}, series = {Nature Communications}, volume = {14}, journal = {Nature Communications}, doi = {10.1038/s41467-023-42634-3}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-358052}, year = {2023}, abstract = {T cell exhaustion is a hallmark of cancer and persistent infections, marked by inhibitory receptor upregulation, diminished cytokine secretion, and impaired cytolytic activity. Terminally exhausted T cells are steadily replenished by a precursor population (Tpex), but the metabolic principles governing Tpex maintenance and the regulatory circuits that control their exhaustion remain incompletely understood. Using a combination of gene-deficient mice, single-cell transcriptomics, and metabolomic analyses, we show that mitochondrial insufficiency is a cell-intrinsic trigger that initiates the functional exhaustion of T cells. At the molecular level, we find that mitochondrial dysfunction causes redox stress, which inhibits the proteasomal degradation of hypoxia-inducible factor 1α (HIF-1α) and promotes the transcriptional and metabolic reprogramming of Tpex cells into terminally exhausted T cells. Our findings also bear clinical significance, as metabolic engineering of chimeric antigen receptor (CAR) T cells is a promising strategy to enhance the stemness and functionality of Tpex cells for cancer immunotherapy.}, language = {en} }