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  1. Article ; Online: Analyzing trogocytosis of T lymphocytes by flow cytometry and confocal microscopy.

    Zink, Alicia / Zenke, Simon / Wiese, Teresa / Beyersdorf, Niklas / Lämmermann, Tim / Rohr, Jan C

    STAR protocols

    2023  Volume 4, Issue 1, Page(s) 102013

    Abstract: Here, we present a protocol to examine the mechanisms underlying the intercellular transfer of transmembrane molecules, termed trogocytosis, and the fate of transferred molecules. We describe the steps needed from T lymphocyte isolation, via co-culture ... ...

    Abstract Here, we present a protocol to examine the mechanisms underlying the intercellular transfer of transmembrane molecules, termed trogocytosis, and the fate of transferred molecules. We describe the steps needed from T lymphocyte isolation, via co-culture with cells expressing the ligand of interest, to cell harvest and subsequent staining for flow cytometry and confocal microscopy. Furthermore, we showcase critical parameters and pitfalls, which allow easy adaptation of the protocol to investigate trogocytosis of various cell surface receptors in different cell types. For complete details on the use and execution of this protocol, please refer to Zink and Rohr.
    MeSH term(s) Flow Cytometry ; T-Lymphocytes ; Trogocytosis ; Microscopy, Confocal ; Coculture Techniques
    Language English
    Publishing date 2023-01-11
    Publishing country United States
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ISSN 2666-1667
    ISSN (online) 2666-1667
    DOI 10.1016/j.xpro.2022.102013
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  2. Article ; Online: Functional Relevance of CTLA4 Variants: an Upgraded Approach to Assess CTLA4-Dependent Transendocytosis by Flow Cytometry.

    Rojas-Restrepo, Jessica / Sindram, Elena / Zenke, Simon / Haberstroh, Hanna / Mitsuiki, Noriko / Gabrysch, Annemarie / Huebscher, Katrin / Posadas-Cantera, Sara / Krausz, Máté / Kobbe, Robin / Rohr, Jan C / Grimbacher, Bodo / Gámez-Díaz, Laura

    Journal of clinical immunology

    2023  Volume 43, Issue 8, Page(s) 2076–2089

    Abstract: Variants of uncertain significance (VUS) in CTLA4 are frequently identified in patients with antibody deficiency or immune dysregulation syndromes including, but not limited to, patients with multi-organ autoimmunity and autoinflammation. However, to ... ...

    Abstract Variants of uncertain significance (VUS) in CTLA4 are frequently identified in patients with antibody deficiency or immune dysregulation syndromes including, but not limited to, patients with multi-organ autoimmunity and autoinflammation. However, to ascertain the diagnosis of CTLA4 insufficiency, the functional relevance of each variant needs to be determined. Currently, various assays have been proposed to assess the functionality of CTLA4 VUS, including the analysis of transendocytosis, the biological function of CTLA4 to capture CD80 molecules from antigen presenting cells. Challenges of this assay include weak fluorescence intensity of the internalized ligand, poor reproducibility, and poor performance upon analyzing thawed cells. In addition, the distinction of pathogenic from non-pathogenic variants and from wild-type CTLA4, and the classification of the different VUS according to its level of CTLA4 dysfunction, would be desirable. We developed a novel CD80-expressing cell line for the evaluation of CD80-transendocytosis and compared it to the published transendocytosis assay. Our approach showed lower inter-assay variability and better robustness regardless the type of starting material (fresh or thawed peripheral mononuclear cells). In addition, receiver operating characteristic analysis showed 100% specificity, avoiding false positive results and allowing for a clear distinction between pathogenic and non-pathogenic variants in CTLA4-variant carriers. With our transendocytosis assay, we assessed the pathogenicity of 24 distinct CTLA4 variants from patients submitted to our diagnostic unit. Significantly impaired transendocytosis was demonstrated for 17 CTLA4 variants, whereas seven variants tested normal. In conclusion, our upgraded transendocytosis assay allows a reliable assessment of newly identified variants in CTLA4.
    MeSH term(s) Humans ; CTLA-4 Antigen/genetics ; Flow Cytometry ; Reproducibility of Results ; Antigen-Presenting Cells ; Autoimmunity
    Chemical Substances CTLA-4 Antigen ; CTLA4 protein, human
    Language English
    Publishing date 2023-09-23
    Publishing country Netherlands
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 779361-3
    ISSN 1573-2592 ; 0271-9142
    ISSN (online) 1573-2592
    ISSN 0271-9142
    DOI 10.1007/s10875-023-01582-9
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  3. Article ; Online: Correction to: Functional Relevance of CTLA4 Variants: an Upgraded Approach to Assess CTLA4‑Dependent Transendocytosis by Flow Cytometry.

    Rojas-Restrepo, Jessica / Sindram, Elena / Zenke, Simon / Haberstroh, Hanna / Mitsuiki, Noriko / Gabrysch, Annemarie / Huebscher, Katrin / Posadas-Cantera, Sara / Krausz, Máté / Kobbe, Robin / Rohr, Jan C / Grimbacher, Bodo / Gamez-Diaz, Laura

    Journal of clinical immunology

    2023  Volume 43, Issue 8, Page(s) 2090

    Language English
    Publishing date 2023-10-09
    Publishing country Netherlands
    Document type Published Erratum
    ZDB-ID 779361-3
    ISSN 1573-2592 ; 0271-9142
    ISSN (online) 1573-2592
    ISSN 0271-9142
    DOI 10.1007/s10875-023-01596-3
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  4. Article ; Online: Differential trafficking of ligands trogocytosed via CD28 versus CTLA4 promotes collective cellular control of co-stimulation.

    Zenke, Simon / Sica, Mauricio P / Steinberg, Florian / Braun, Julia / Zink, Alicia / Gavrilov, Alina / Hilger, Alexander / Arra, Aditya / Brunner-Weinzierl, Monika / Elling, Roland / Beyersdorf, Niklas / Lämmermann, Tim / Smulski, Cristian R / Rohr, Jan C

    Nature communications

    2022  Volume 13, Issue 1, Page(s) 6459

    Abstract: Intercellular communication is crucial for collective regulation of cellular behaviors. While clustering T cells have been shown to mutually control the production of key communication signals, it is unclear whether they also jointly regulate their ... ...

    Abstract Intercellular communication is crucial for collective regulation of cellular behaviors. While clustering T cells have been shown to mutually control the production of key communication signals, it is unclear whether they also jointly regulate their availability and degradation. Here we use newly developed reporter systems, bioinformatic analyses, protein structure modeling and genetic perturbations to assess this. We find that T cells utilize trogocytosis by competing antagonistic receptors to differentially control the abundance of immunoregulatory ligands. Specifically, ligands trogocytosed via CD28 are shuttled to the T cell surface, enabling them to co-stimulate neighboring T cells. In contrast, CTLA4-mediated trogocytosis targets ligands for degradation. Mechanistically, this fate separation is controlled by different acid-sensitivities of receptor-ligand interactions and by the receptor intracellular domains. The ability of CD28 and CTLA4 to confer different fates to trogocytosed ligands reveals an additional layer of collective regulation of cellular behaviors and promotes the robustness of population dynamics.
    MeSH term(s) CD28 Antigens/genetics ; CD28 Antigens/metabolism ; CTLA-4 Antigen/genetics ; Ligands ; Abatacept ; Antigens, CD ; Immunoconjugates
    Chemical Substances CD28 Antigens ; CTLA-4 Antigen ; Ligands ; Abatacept (7D0YB67S97) ; Antigens, CD ; Immunoconjugates
    Language English
    Publishing date 2022-10-29
    Publishing country England
    Document type Journal Article
    ZDB-ID 2553671-0
    ISSN 2041-1723 ; 2041-1723
    ISSN (online) 2041-1723
    ISSN 2041-1723
    DOI 10.1038/s41467-022-34156-1
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  5. Article ; Online: Quorum Regulation via Nested Antagonistic Feedback Circuits Mediated by the Receptors CD28 and CTLA-4 Confers Robustness to T Cell Population Dynamics.

    Zenke, Simon / Palm, Margriet M / Braun, Julia / Gavrilov, Alina / Meiser, Philippa / Böttcher, Jan P / Beyersdorf, Niklas / Ehl, Stephan / Gerard, Audrey / Lämmermann, Tim / Schumacher, Ton N / Beltman, Joost B / Rohr, Jan C

    Immunity

    2020  Volume 52, Issue 2, Page(s) 313–327.e7

    Abstract: T cell responses upon infection display a remarkably reproducible pattern of expansion, contraction, and memory formation. If the robustness of this pattern builds entirely on signals derived from other cell types or if activated T cells themselves ... ...

    Abstract T cell responses upon infection display a remarkably reproducible pattern of expansion, contraction, and memory formation. If the robustness of this pattern builds entirely on signals derived from other cell types or if activated T cells themselves contribute to the orchestration of these population dynamics-akin to bacterial quorum regulation-is unclear. Here, we examined this question using time-lapse microscopy, genetic perturbation, bioinformatic predictions, and mathematical modeling. We found that ICAM-1-mediated cell clustering enabled CD8
    MeSH term(s) Animals ; B7-1 Antigen/metabolism ; B7-2 Antigen/metabolism ; CD28 Antigens/metabolism ; CD8-Positive T-Lymphocytes/cytology ; CD8-Positive T-Lymphocytes/immunology ; CD8-Positive T-Lymphocytes/metabolism ; CTLA-4 Antigen/metabolism ; Cell Communication ; Cell Count ; Cell Line ; Cell Survival ; Cell Tracking ; Dendritic Cells/immunology ; Intercellular Adhesion Molecule-1/metabolism ; Interleukin-2/metabolism ; Lymphocyte Activation ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Models, Theoretical
    Chemical Substances B7-1 Antigen ; B7-2 Antigen ; CD28 Antigens ; CTLA-4 Antigen ; Cd86 protein, mouse ; Ctla4 protein, mouse ; Interleukin-2 ; Intercellular Adhesion Molecule-1 (126547-89-5)
    Language English
    Publishing date 2020-02-11
    Publishing country United States
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 1217235-2
    ISSN 1097-4180 ; 1074-7613
    ISSN (online) 1097-4180
    ISSN 1074-7613
    DOI 10.1016/j.immuni.2020.01.018
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  6. Article: Cytomegalovirus subverts macrophage identity

    Baasch, Sebastian / Giansanti, Piero / Kolter, Julia / Riedl, André / Forde, Aaron James / Runge, Solveig / Zenke, Simon / Elling, Roland / Halenius, Anne / Brabletz, Simone / Hengel, Hartmut / Kuster, Bernhard / Brabletz, Thomas / Cicin-Sain, Luka / Arens, Ramon / Vlachos, Andreas / Rohr, Jan Christopher / Stemmler, Marc Philippe / Kopf, Manfred /
    Ruzsics, Zsolt / Henneke, Philipp

    Cell. 2021 July 08, v. 184, no. 14

    2021  

    Abstract: Cytomegaloviruses (CMVs) have co-evolved with their mammalian hosts for millions of years, leading to remarkable host specificity and high infection prevalence. Macrophages, which already populate barrier tissues in the embryo, are the predominant immune ...

    Abstract Cytomegaloviruses (CMVs) have co-evolved with their mammalian hosts for millions of years, leading to remarkable host specificity and high infection prevalence. Macrophages, which already populate barrier tissues in the embryo, are the predominant immune cells at potential CMV entry sites. Here we show that, upon CMV infection, macrophages undergo a morphological, immunophenotypic, and metabolic transformation process with features of stemness, altered migration, enhanced invasiveness, and provision of the cell cycle machinery for viral proliferation. This complex process depends on Wnt signaling and the transcription factor ZEB1. In pulmonary infection, mouse CMV primarily targets and reprograms alveolar macrophages, which alters lung physiology and facilitates primary CMV and secondary bacterial infection by attenuating the inflammatory response. Thus, CMV profoundly perturbs macrophage identity beyond established limits of plasticity and rewires specific differentiation processes, allowing viral spread and impairing innate tissue immunity.
    Keywords Cytomegalovirus ; bacterial infections ; cell cycle ; coevolution ; host specificity ; immunity ; inflammation ; lungs ; macrophages ; mice ; physiology ; plasticity ; transcription factors
    Language English
    Dates of publication 2021-0708
    Size p. 3774-3793.e25.
    Publishing place Elsevier Inc.
    Document type Article
    ZDB-ID 187009-9
    ISSN 1097-4172 ; 0092-8674
    ISSN (online) 1097-4172
    ISSN 0092-8674
    DOI 10.1016/j.cell.2021.05.009
    Database NAL-Catalogue (AGRICOLA)

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  7. Article ; Online: Cytomegalovirus subverts macrophage identity.

    Baasch, Sebastian / Giansanti, Piero / Kolter, Julia / Riedl, André / Forde, Aaron James / Runge, Solveig / Zenke, Simon / Elling, Roland / Halenius, Anne / Brabletz, Simone / Hengel, Hartmut / Kuster, Bernhard / Brabletz, Thomas / Cicin-Sain, Luka / Arens, Ramon / Vlachos, Andreas / Rohr, Jan Christopher / Stemmler, Marc Philippe / Kopf, Manfred /
    Ruzsics, Zsolt / Henneke, Philipp

    Cell

    2021  Volume 184, Issue 14, Page(s) 3774–3793.e25

    Abstract: Cytomegaloviruses (CMVs) have co-evolved with their mammalian hosts for millions of years, leading to remarkable host specificity and high infection prevalence. Macrophages, which already populate barrier tissues in the embryo, are the predominant immune ...

    Abstract Cytomegaloviruses (CMVs) have co-evolved with their mammalian hosts for millions of years, leading to remarkable host specificity and high infection prevalence. Macrophages, which already populate barrier tissues in the embryo, are the predominant immune cells at potential CMV entry sites. Here we show that, upon CMV infection, macrophages undergo a morphological, immunophenotypic, and metabolic transformation process with features of stemness, altered migration, enhanced invasiveness, and provision of the cell cycle machinery for viral proliferation. This complex process depends on Wnt signaling and the transcription factor ZEB1. In pulmonary infection, mouse CMV primarily targets and reprograms alveolar macrophages, which alters lung physiology and facilitates primary CMV and secondary bacterial infection by attenuating the inflammatory response. Thus, CMV profoundly perturbs macrophage identity beyond established limits of plasticity and rewires specific differentiation processes, allowing viral spread and impairing innate tissue immunity.
    MeSH term(s) Animals ; Antigen Presentation ; Bystander Effect ; Cell Cycle ; Cell Line, Transformed ; Cellular Reprogramming ; Cytomegalovirus/pathogenicity ; Cytomegalovirus/physiology ; Cytomegalovirus/ultrastructure ; Cytomegalovirus Infections/immunology ; Cytomegalovirus Infections/virology ; Green Fluorescent Proteins/metabolism ; Lung/pathology ; Macrophages, Alveolar/immunology ; Macrophages, Alveolar/ultrastructure ; Macrophages, Alveolar/virology ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Phenotype ; Stem Cells/pathology ; Virus Replication/physiology ; Wnt Signaling Pathway ; Mice
    Chemical Substances Green Fluorescent Proteins (147336-22-9)
    Language English
    Publishing date 2021-06-10
    Publishing country United States
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 187009-9
    ISSN 1097-4172 ; 0092-8674
    ISSN (online) 1097-4172
    ISSN 0092-8674
    DOI 10.1016/j.cell.2021.05.009
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  8. Article ; Online: Early-onset Evans syndrome, immunodeficiency, and premature immunosenescence associated with tripeptidyl-peptidase II deficiency.

    Stepensky, Polina / Rensing-Ehl, Anne / Gather, Ruth / Revel-Vilk, Shoshana / Fischer, Ute / Nabhani, Schafiq / Beier, Fabian / Brümmendorf, Tim H / Fuchs, Sebastian / Zenke, Simon / Firat, Elke / Pessach, Vered Molho / Borkhardt, Arndt / Rakhmanov, Mirzokhid / Keller, Bärbel / Warnatz, Klaus / Eibel, Hermann / Niedermann, Gabriele / Elpeleg, Orly /
    Ehl, Stephan

    Blood

    2014  Volume 125, Issue 5, Page(s) 753–761

    Abstract: Autoimmune cytopenia is a frequent manifestation of primary immunodeficiencies. Two siblings presented with Evans syndrome, viral infections, and progressive leukopenia. DNA available from one patient showed a homozygous frameshift mutation in ... ...

    Abstract Autoimmune cytopenia is a frequent manifestation of primary immunodeficiencies. Two siblings presented with Evans syndrome, viral infections, and progressive leukopenia. DNA available from one patient showed a homozygous frameshift mutation in tripeptidyl peptidase II (TPP2) abolishing protein expression. TPP2 is a serine exopeptidase involved in extralysosomal peptide degradation. Its deficiency in mice activates cell death programs and premature senescence. Similar to cells from naïve, uninfected TPP2-deficient mice, patient cells showed increased major histocompatibility complex I expression and most CD8(+) T-cells had a senescent CCR7-CD127(-)CD28(-)CD57(+) phenotype with poor proliferative responses and enhanced staurosporine-induced apoptosis. T-cells showed increased expression of the effector molecules perforin and interferon-γ with high expression of the transcription factor T-bet. Age-associated B-cells with a CD21(-) CD11c(+) phenotype expressing T-bet were increased in humans and mice, combined with antinuclear antibodies. Moreover, markers of senescence were also present in human and murine TPP2-deficient fibroblasts. Telomere lengths were normal in patient fibroblasts and granulocytes, and low normal in lymphocytes, which were compatible with activation of stress-induced rather than replicative senescence programs. TPP2 deficiency is the first primary immunodeficiency linking premature immunosenescence to severe autoimmunity. Determination of senescent lymphocytes should be part of the diagnostic evaluation of children with refractory multilineage cytopenias.
    MeSH term(s) Aging/immunology ; Aminopeptidases/deficiency ; Aminopeptidases/genetics ; Aminopeptidases/immunology ; Anemia, Hemolytic, Autoimmune/complications ; Anemia, Hemolytic, Autoimmune/genetics ; Anemia, Hemolytic, Autoimmune/immunology ; Anemia, Hemolytic, Autoimmune/pathology ; Animals ; Apoptosis ; Base Sequence ; CD8-Positive T-Lymphocytes/immunology ; CD8-Positive T-Lymphocytes/metabolism ; CD8-Positive T-Lymphocytes/pathology ; Child ; Child, Preschool ; Consanguinity ; Dipeptidyl-Peptidases and Tripeptidyl-Peptidases/deficiency ; Dipeptidyl-Peptidases and Tripeptidyl-Peptidases/genetics ; Dipeptidyl-Peptidases and Tripeptidyl-Peptidases/immunology ; Female ; Fibroblasts/immunology ; Fibroblasts/metabolism ; Fibroblasts/pathology ; Frameshift Mutation ; Gene Expression ; Humans ; Immunologic Deficiency Syndromes/complications ; Immunologic Deficiency Syndromes/genetics ; Immunologic Deficiency Syndromes/immunology ; Immunologic Deficiency Syndromes/pathology ; Male ; Mice ; Mice, Knockout ; Molecular Sequence Data ; Perforin/genetics ; Perforin/immunology ; Serine Endopeptidases/deficiency ; Serine Endopeptidases/genetics ; Serine Endopeptidases/immunology ; Siblings ; T-Box Domain Proteins/genetics ; T-Box Domain Proteins/immunology ; T-Lymphocytes/immunology ; T-Lymphocytes/metabolism ; T-Lymphocytes/pathology ; Thrombocytopenia/complications ; Thrombocytopenia/genetics ; Thrombocytopenia/immunology ; Thrombocytopenia/pathology
    Chemical Substances T-Box Domain Proteins ; T-box transcription factor TBX21 ; Perforin (126465-35-8) ; Aminopeptidases (EC 3.4.11.-) ; Dipeptidyl-Peptidases and Tripeptidyl-Peptidases (EC 3.4.14.-) ; tripeptidyl-peptidase 2 (EC 3.4.14.10) ; Serine Endopeptidases (EC 3.4.21.-)
    Language English
    Publishing date 2014-11-20
    Publishing country United States
    Document type Case Reports ; Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 80069-7
    ISSN 1528-0020 ; 0006-4971
    ISSN (online) 1528-0020
    ISSN 0006-4971
    DOI 10.1182/blood-2014-08-593202
    Database MEDical Literature Analysis and Retrieval System OnLINE

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